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Abstract:

Compositions, methods, and kits for detecting one or more species of RNA
molecules are disclosed. In one embodiment, a first adaptor and a second
adaptor are ligated to the RNA molecule using a polypeptide comprising
double-strand specific RNA ligase activity, without an intervening
purification step. The ligated product is reverse transcribed, then at
least some of the ribonucleosides in the reverse transcription product
are removed. Primers are added and amplified products are generated. In
certain embodiments, the sequence of at least part of at least one
species of amplified product is determined and at least part of the
corresponding RNA molecule is determined. In some embodiments, at least
some of the amplified product species are detected, directly or
indirectly, allowing the presence and/or quantity of the RNA molecule of
interest to be determined.

Claims:

1. A method for detecting a RNA molecule in a sample, comprising:
combining the sample with at least one first adaptor, at least one second
adaptor, and a polypeptide comprising double-strand specific RNA ligase
activity to form a ligation reaction composition in which the at least
one first adaptor and the at least one second adaptor are ligated to the
RNA molecule of the sample to form a ligated product in the same ligation
reaction composition, wherein the at least one first adaptor comprises: a
first oligonucleotide having a length of 10 to 60 nucleotides and
comprising at least two ribonucleosides on the 3'-end, and a second
oligonucleotide comprising a nucleotide sequence substantially
complementary to the first oligonucleotide and further comprising a
single-stranded 5' portion of 1 to 8 nucleotides when the first
oligonucleotide and the second oligonucleotide are duplexed, wherein the
at least one second adaptor comprises: a third oligonucleotide having a
length of 10 to 60 nucleotides and comprising a 5' phosphate group, and a
fourth oligonucleotide comprising a nucleotide sequence substantially
complementary to the third oligonucleotide and further comprising a
single-stranded 3' portion of 1 to 8 nucleotides when the third
oligonucleotide and the fourth oligonucleotide are duplexed, wherein the
single-stranded portions independently have a degenerate nucleotide
sequence, or a sequence that is complementary to a portion of the RNA
molecule, wherein the first and third oligonucleotides have a different
nucleotide sequence; wherein the RNA molecule hybridizes with the
single-stranded portion of the at least one first adaptor and the
single-stranded portion of the at least one second adaptor; and detecting
the RNA molecule of the ligated product or a surrogate thereof.

2. The method of claim 1 wherein detecting the RNA molecule or a
surrogate thereof comprises: combining the ligated product with i) a
RNA-directed DNA polymerase, ii) a DNA polymerase comprising DNA
dependent DNA polymerase activity and RNA dependent DNA polymerase
activity, or iii) a RNA-directed DNA polymerase and a DNA-directed DNA
polymerase, reverse transcribing the ligated product to form a reverse
transcribed product, combining the amplification template with at least
one forward primer, at least one reverse primer, and a DNA-directed DNA
polymerase when the ligated product is combined as in i), to form an
amplification reaction composition, cycling the amplification reaction
composition to form at least one amplified product, and determining the
sequence of at least part of the amplified product, thereby detecting the
RNA molecule.

5. The method of claim 1, wherein the single-stranded portion of the
first adaptor, the single-stranded portion of the second adaptor, or the
single-stranded portion of the first adaptor and the single-stranded
portion of the second adaptor, comprise degenerate sequences.

6. The method of claim 1, wherein the first oligonucleotide comprises at
least fifteen ribonucleosides.

7. The method of claim 1, wherein the second adaptor comprises at least
one reporter group.

8. The method of claim 2, wherein the at least one amplified product
comprises an identification sequence.

9. The method of claim 8, wherein at least one of the at least one
forward primer, at least one of the at least one reverse primer, or both,
comprise an identification sequence.

10. The method of claim 8, wherein at least one first adaptor, at least
one second adaptor, or at least one first adaptor and at least one second
adaptor comprises an identification sequence.

11. The method of claim 1, wherein the RNA molecule is a small non-coding
RNA.

12. The method of claim 1, wherein the sample comprises a plurality of
RNA fragments and the detecting comprises determining an expression
profile.

13. The method of claim 12, wherein a concentration of at least one
species of RNA molecule is depleted prior to forming the ligated product.

14. The method of claim 12, wherein the sample comprises a plurality of
messenger RNA (mRNA) fragments and the detecting comprises determining an
expression profile.

15. The method of claim 1, wherein the detecting comprises sequencing at
least part of an amplified product, wherein the sequencing comprises a
massive parallel signature sequencing reaction, hybridizing at least one
amplified product to a microarray, or cloning at least one amplified
product into a sequencing vector.

16. The method of claim 1, wherein the single-stranded portion of the
first adaptor, the single-stranded portion of the second adaptor, or the
single-stranded portion of the first adaptor and the single-stranded
portion of the second adaptor comprise a sequence-specific region to
selectively hybridize with a corresponding RNA molecule.

17. A method for detecting an RNA molecule, comprising: combining the RNA
molecule with at least one first adaptor, at least one second adaptor,
and a double-strand specific ligase to form a ligation reaction
composition, wherein the at least one first adaptor comprises a first
oligonucleotide comprising at least two ribonucleosides on the 3'-end and
a second oligonucleotide that comprises a single-stranded portion when
the first oligonucleotide and the second oligonucleotide are hybridized
together, and wherein the at least one second adaptor comprises a third
oligonucleotide that comprises a 5' phosphate group and a fourth
oligonucleotide that comprises a single-stranded portion when the third
oligonucleotide and the fourth oligonucleotide are hybridized together,
ligating the at least one first adaptor and the at least one second
adaptor to the RNA molecule to form a ligated product, wherein the first
adaptor and the second adaptor are ligated to the RNA molecule in the
same ligation reaction composition, combining the ligated product with an
RNA-directed DNA polymerase, reverse transcribing the ligated product to
form a reverse transcribed product, combining the amplification template
with at least one forward primer, at least one reverse primer, and a
DNA-directed DNA polymerase to form an amplification reaction
composition, cycling the amplification reaction composition to form an
amplified product, and determining the sequence of at least part of the
amplified product, thereby detecting the RNA molecule.

18. The method of claim 17, wherein the single-stranded portion of the
first adaptor, the single-stranded portion of the second adaptor, or the
single-stranded portion of the first adaptor and the single-stranded
portion of the second adaptor, comprise degenerate sequences.

19. The method of claim 17, wherein the at least two ribonucleosides of
the first oligonucleotide comprises at least fifteen ribonucleosides.

20. The method of claim 17, wherein the second adaptor comprises at least
one reporter group.

21. The method of claim 17, wherein at least one of the amplified
products comprises an identification sequence.

22-38. (canceled)

Description:

CROSS-RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.
13/463,758 filed May 3, 2012, which is a continuation of U.S. application
Ser. No. 12/835,869 filed Jul. 14, 2010 (now U.S. Pat. No. 8,192,941),
which is a continuation of, and claims priority to, International Patent
Application No. PCT/US2009/030822, having an international filing date of
Jan. 13, 2009, which application claims the benefit of U.S. Provisional
Application No. 61/020,913 filed Jan. 14, 2008, U.S. Provisional
Application No. 61/039,460 filed Mar. 26, 2008 and U.S. Application No.
61/047,549 filed Apr. 24, 2008. Each application is incorporated by
reference herein in its entirety.

FIELD

[0002] The present teachings generally relate to methods, reagents, and
kits for detecting, amplifying, and quantifying ribonucleic acid (RNA),
including but not limited to coding RNA and non-coding RNA (ncRNA).

INTRODUCTION

[0003] Analysis of genome expression patterns provides valuable insight
into the role of differential expression in a wide variety of biological
processes, including but not limited to, various disease states. Such
analysis, whether mRNA-based gene expression or small non-coding
RNA-based expression analysis, is becoming a rapidly expanding avenue of
investigation in many disciplines in the biological sciences. Small
non-coding RNA discovery is also an area of great scientific and medical
interest. It is believed that by knowing what parts of the genome are
transcribed when and why, a better understanding of many complex and
inter-related biological processes may be obtained.

[0004] Small non-coding RNAs are rapidly emerging as significant effectors
of gene regulation in a multitude of organisms spanning the evolutionary
spectrum. Animals, plants and fungi contain several distinct classes of
small RNAs; including without limitation, miRNAs, siRNAs, piRNAs, and
rasiRNAs. These tiny gene expression modulators typically fall within the
size range of ˜18-40 nt in length, however their effect on cellular
processes is profound. They have been shown to play critical roles in
developmental timing and cell fate mechanisms, tumor progression,
neurogenesis, transposon silencing, viral defense and many more. They
function in gene regulation by binding to their targets and negatively
effecting gene expression by a variety of mechanisms including
heterochromatin modification, translational inhibition, mRNA decay and
even nascent peptide turnover mechanisms. Therefore, identification of
the small RNAs in a given sample can greatly facilitate gene expression
analysis.

[0005] Some small RNAs are produced from defined locations within the
genome. MicroRNAs are such a class; they are typically transcribed by RNA
polymerase II from polycistronic gene clusters or can also be generated
from pre-mRNA introns. Thus far several thousand unique miRNA sequences
are known. Other classes of small RNAs, such as piRNAs or endogenous
siRNA, are not typically transcribed from a defined locus in the genome.
Instead, they are generated in response to events such as viral
infections or retrotransposon expression and serve to silence these
`foreign` sequences that would otherwise result in serious detriment to
the cell. Descriptions of ncRNA can be found in, among other places,
Eddy, Nat. Rev. Genet. 2:919-29, 2001; Mattick and Makunin, Human Mol.
Genet. 15:R17-29, 2006; Hannon et al., Cold Springs Harbor Sympos. Quant.
Biol. LXXI:551-64, 2006. Sequencing the entire population of small RNAs
in a sample provides a direct method to identify and even profile all
classes of these RNAs at one time.

SUMMARY

[0006] The present teachings are directed to methods, reagents, and kits
for detecting and quantitating: (i) small RNA molecules, also referred to
as untranslated functional RNA, non-coding RNA (ncRNA), and small
non-messenger RNA (snmRNA); and (ii) coding RNA, which may or may not be
fragmented and/or fractionated by methods known in the art.

[0007] According to certain disclosed methods, a ligation reaction
composition is formed comprising at least one RNA molecule to be
detected, at least one first adaptor, at least one second adaptor, and a
double-strand specific RNA ligase. The first adaptor comprises a first
oligonucleotide comprising at least two ribonucleosides on the 3'-end and
a second oligonucleotide that comprises a single-stranded portion when
the first oligonucleotide and the second oligonucleotide are hybridized
together. The second adaptor comprises a third oligonucleotide that
comprises a 5' phosphate group and a fourth oligonucleotide that
comprises a single-stranded portion when the third oligonucleotide and
the fourth oligonucleotide are hybridized together. A first adaptor and a
second adaptor are ligated to an RNA molecule in the ligation reaction
composition by the double-strand specific RNA ligase to form a ligated
product. The first adaptor and the second adaptor anneal with the RNA
molecule in a directional manner due to their structure and each adaptor
is ligated simultaneously or nearly simultaneously to the RNA molecule
with which it is annealed, rather than sequentially (for example, when a
second adaptor and the RNA molecule are combined with a ligase and the
second adaptor is ligated to the 3' end of the RNA molecule, then
subsequently a first adaptor is combined with the ligated RNA
molecule-second adaptor and the first adaptor is then ligated to the 5'
end of the RNA molecule-second adaptor, with an intervening purification
step between ligating the second adaptor to the RNA molecule and ligating
the first adaptor to the RNA molecule, see, e.g., Elbashir et al, Genes
and Development 15: 188-200, 2001; Berezikov et al., Nat. Genet. Supp.
38: S2-S7, 2006). It is to be appreciated that the order in which
components are added to the ligation reaction composition is not limiting
and that the components may be added in any order. It is also to be
appreciated that during the process of adding components, an adaptor may
be ligated with a corresponding RNA molecule in the presence of a ligase
before all of the components of the reaction composition are added, for
example but without limitation, a second adaptor may be ligated with a
corresponding RNA molecule in the presence of a ligase before the first
adaptors are added, and that such reactions are within the intended scope
of the current teachings, provided there is not a purification procedure
between the time one adaptor is ligated to the RNA molecule and the time
the other adaptor is ligated to the RNA molecule. An RNA-directed DNA
polymerase (sometimes referred to as an RNA-dependent DNA polymerase) is
combined with the ligated product to form reaction mixture, which is
incubated under conditions suitable for a reverse transcribed product.
The reverse transcribed product is combined with a ribonuclease,
typically ribonuclease H(RNase H), and at least some of the
ribonucleosides are digested from the reverse transcribed product to form
an amplification template.

[0008] The amplification template is combined with at least one forward
primer, at least one reverse primer, and a DNA-directed DNA polymerase
(sometimes referred to as a DNA-dependent DNA polymerase) to form an
amplification reaction composition. The amplification reaction
composition is thermocycled under conditions suitable to allow amplified
products to be generated. In some embodiments, at least one species of
amplified product is detected. In some embodiments, a reporter probe
and/or a nucleic acid dye is used to indirectly detect the presence of at
least one of the RNA species in the sample. In certain embodiments, an
amplification reaction composition further comprises a reporter probe,
for example but not limited to a TaqMan® probe, molecular beacon,
Scorpion® primer or the like, or a nucleic acid dye, for example but
not limited to, SYBR® Green or other nucleic acid binding dye or
nucleic acid intercalating dye. In certain embodiments of the current
teachings, detecting comprises a real-time or end-point detection
technique, including without limitation, quantitative PCR. In some
embodiments, the sequence of at least part of the amplified product is
determined, which allows the corresponding RNA molecule to be identified.
In some embodiments, a library of amplified products comprising a
library-specific nucleotide sequence is generated from the RNA molecules
in a starting material, wherein at least some of the amplified product
species share a library-specific identifier, for example but not limited
to a library-specific nucleotide sequence, including without limitation,
a barcode sequence or a hybridization tag, or a common marker or affinity
tag. In some embodiments, two or more libraries are combined and
analyzed, then the results are deconvoluted based on the library-specific
identifier.

[0009] According to certain disclosed methods, only one polymerase, a DNA
polymerase comprising both DNA-directed DNA polymerase activity and
RNA-directed DNA polymerase activity, is employed in the reverse
transcription reaction composition and no additional polymerase is used.
In other method embodiments, both an RNA-directed DNA polymerase and a
DNA-directed DNA polymerase are added to the reverse transcription
reaction composition and no additional polymerase is added to the
amplification reaction composition.

[0010] In some embodiments, a method for detecting a RNA molecule in a
sample comprises combining the sample with at least one first adaptor, at
least one second adaptor, and a polypeptide comprising double-strand
specific RNA ligase activity to form a ligation reaction composition in
which the at least one first adaptor and the at least one second adaptor
are ligated to the RNA molecule of the sample to form a ligated product
in the same ligation reaction composition, and detecting the RNA molecule
of the ligated product or a surrogate thereof. In some embodiments, the
at least one first adaptor comprises a first oligonucleotide having a
length of 10 to 60 nucleotides and comprising at least two
ribonucleosides on the 3'-end, and a second oligonucleotide comprising a
nucleotide sequence substantially complementary to the first
oligonucleotide and further comprising a single-stranded 5' portion of 1
to 8 nucleotides when the first oligonucleotide and the second
oligonucleotide are duplexed. In some embodiments, the at least one
second adaptor comprises a third oligonucleotide having a length of 10 to
60 nucleotides and comprising a 5' phosphate group, and a fourth
oligonucleotide comprising a nucleotide sequence substantially
complementary to the third oligonucleotide and further comprising a
single-stranded 3' portion of 1 to 8 nucleotides when the third
oligonucleotide and the fourth oligonucleotide are duplexed. In some
embodiments, the single-stranded portions independently have a degenerate
nucleotide sequence, or a sequence that is complementary to a portion of
the RNA molecule. In some embodiments, the first and third
oligonucleotides have a different nucleotide sequence. In the ligaton
reaction composition, the RNA molecule to be detected hybridizes with the
single-stranded portion of the at least one first adaptor and the
single-stranded portion of the at least one second adaptor.

[0011] In some embodiments, detecting the RNA molecule or a surrogate
thereof comprises combining the ligated product with i) a RNA-directed
DNA polymerase, ii) a DNA polymerase comprising DNA dependent DNA
polymerase activity and RNA dependent DNA polymerase activity, or iii) a
RNA-directed DNA polymerase and a DNA-directed DNA polymerase; reverse
transcribing the ligated product to form a reverse transcribed product;
digesting at least some of the ribonucleosides from the reverse
transcribed product with ribonuclease H to form an amplification
template; combining the amplification template with at least one forward
primer, at least one reverse primer, and a DNA-directed DNA polymerase
when the ligated product is combined as in i), to form an amplification
reaction composition; cycling the amplification reaction composition to
form at least one amplified product, and determining the sequence of at
least part of the amplified product, thereby detecting the RNA molecule.

[0012] In some embodiments, a method for generating an RNA library
comprises combining a multiplicity of different RNA molecules with a
multiplicity of first adaptor species, a multiplicity of second adaptor
species, and a double-strand specific RNA ligase to form a ligation
reaction composition, wherein the at least one first adaptor comprises a
first oligonucleotide comprising at least two ribonucleosides on the
3'-end and a second oligonucleotide that comprises a single-stranded
portion when the first oligonucleotide and the second oligonucleotide are
hybridized together, and wherein the at least one second adaptor
comprises a third oligonucleotide that comprises a 5' phosphate group and
a fourth oligonucleotide that comprises a single-stranded portion when
the third oligonucleotide and the fourth oligonucleotide are hybridized
together and ligating the at least one first adaptor and the at least one
second adaptor to the RNA molecule to form a multiplicity of different
ligated product species, wherein the first adaptor and the second adaptor
are ligated to the RNA molecule in the same ligation reaction
composition. The method further comprises combining the multiplicity of
ligated product species with an RNA-directed DNA polymerase, reverse
transcribing at least some of the multiplicity of ligated product species
to form a multiplicity of reverse transcribed product species, digesting
at least some of the ribonucleosides from at least some of the
multiplicity of reverse transcribed products with a ribonuclease H(RNase
H) to form a multiplicity of amplification template species, combining
the multiplicity of amplification template species with at least one
forward primer, at least one reverse primer, and a DNA-directed DNA
polymerase to form an amplification reaction composition, and cycling the
amplification reaction composition to form a library comprising a
multiplicity of amplified product species, wherein at least some of the
amplified product species comprise an identification sequence that is
common to at least some of the other amplified product species in the
library.

[0013] Kits for performing certain of the instant methods are also
disclosed. These and other features of the present teachings are set
forth herein.

DRAWINGS

[0014] The skilled artisan will understand that the drawings, described
below, are for illustration purposes only. These figures are not intended
to limit the scope of the present teachings in any way.

[0015] FIG. 1 provides a schematic overview of various exemplary method
embodiments of the current teachings.

[0016]FIG. 2A-FIG. 2B: FIG. 2A schematically depicts an exemplary first
adaptor 21 and an exemplary second adaptor 22; FIG. 2B schematically
depicts the exemplary first and second adaptors shown in FIG. 1A
directionally annealed to an exemplary RNA molecule 23. The ligation
junction for the first adaptor 21 and the 5' end of the RNA molecule 23
is shown by arrow 24 and the ligation junction for the second adaptor 22
and the 3' end of the RNA molecule 23 is shown by arrow 25. Open
rectangles depict RNA sequence such as for 21A and 23. Horizontal solid
lines depict DNA sequence such as for 21B and 22A.

[0017]FIG. 3 provides a schematic overview of an exemplary embodiment of
the current teachings. A population of small RNA molecules 33 is combined
with a first adaptor 31 and a second adaptor 32 and Rnl2 ligase to form
ligated product 34. Unannealed adaptors and/or undesired annealed
byproduct molecules 35 may also be present in the ligated reaction
composition. The reaction composition is combined with an RNA-directed
DNA polymerase to generate reverse transcribed product 36, which
composition is then combined with ribonuclease H to generate
amplification template 38. The amplification template 38 is combined with
a DNA-directed DNA polymerase, a forward primer 310 and a reverse primer
311 to form an amplification reaction composition. In this illustrative
embodiment, the reverse primer further comprises an identification
sequence 312, sometimes referred to as a "bar code" sequence.

[0018]FIG. 4 provides a schematic overview of an exemplary embodiment of
the current teachings. In this illustrative embodiment, the amplified
product is gel purified (Gel Purif.) and comprises an insert sequence
(shown by a curved bracket in FIG. 4), a first primer region (shown as P1
in FIG. 4), and a second primer region (shown as P2 in FIG. 4) that
includes a bar code or identification sequence (shown as be in FIG. 4).

[0019] FIG. 5 depicts an electropherogram of the exemplary amplified
products generated as described in Example 1.

[0035] Lane 9: 5 units each of bacteriophage T4 RNA ligase I and
bacteriophage T4 DNA ligase, no RT; and

[0036] Lane 10: no ligase, 200 U RT.

[0037]FIG. 7A depicts an electropherogram of exemplary ligated products
(shown by arrow annotated double ligation) generated in a ligation
reaction composition comprising various first adaptors, various second
adaptors, or combinations of various first adaptors and various second
adaptors, with a multiplicity of different miRNA molecules comprised of
approximately equimolar concentrations of about five hundred different
species of synthetic miRNA molecules and RNA ligase 2 of bacteriophage
T4, as described in Example 4. The numbers 4, 6, and 8 across the top of
FIG. 7A correspond to the number of degenerate nucleotide sequences on
the second oligonucleotide of each of the first adaptors and the fourth
oligonucleotide of each of the second adaptors in the reaction
composition (shown as N in FIG. 7B).

[0038] X-axis:

[0039] T3-4 indicates that the corresponding ligation products were
generated in a ligation composition comprising only first adaptors
comprising the structure T3:27 N (see FIG. 7B), where in this case N
equals 4 degenerate nucleotides;

[0040] T3-6 indicates that the corresponding ligation products were
generated in a ligation reaction composition containing only first
adaptors comprising the structure T3:27 N, where N in this case equals 6
degenerate nucleotides;

[0041] T3-8 indicates that the corresponding ligation products were
generated in a ligation reaction containing only first adaptors
comprising the structure T3:27 N, where N in this case equals 8
degenerate nucleotides;

[0042] T7-4 indicates that the corresponding ligation products were
generated in a ligation reaction with only second adaptors comprising the
structure T7:N 28 (see FIG. 7B), where N in this case equals 4 degenerate
nucleotides;

[0043] T7-6 indicates that the corresponding ligation products were
generated in a ligation reaction with only second adaptors comprising the
structure T7:N 28 (see FIG. 7B), where N in this case equals 6 degenerate
nucleotides;

[0044] T7-8 indicates that the corresponding ligation products were
generated in a ligation reaction with only second adaptors comprising the
structure T7:N 28, where N in this case equals 8 degenerate nucleotides;

[0045] T3-4+T7-4 indicates that the corresponding ligation products were
generated in a ligation reaction with first adaptors comprising the
structure T3:27 N and second adaptors comprising the structure T7:N 28,
where N in this case equals 4 degenerate nucleotides in all species of
both adaptors;

[0046] T3-6+T7-6 indicates that the corresponding ligation products were
generated in a ligation reaction with first adaptors comprising the
structure T3:27 N and second adaptors comprising the structure T7:N 28,
where N in this case equals 6 degenerate nucleotides in all species of
both adaptors; and

[0047] T3-8+T7-8 indicates that the corresponding ligation products were
generated in a ligation reaction with first adaptors comprising the
structure T3:27 N and second adaptors comprising the structure T7:N 28,
where N in this case equals 8 degenerate nucleotides in all species of
both adaptors.

[0048] The number 27 refers to the length of the first oligonucleotide of
the first adaptor and the number 28 refers to the length of the third
nucleotide of the second adaptor.

[0049]FIG. 7B schematically depicts exemplary first and second adaptors
of the current teachings, where N represents a series of degenerate
nucleosides on the lower strand of either the exemplary first adaptor or
the second adaptor, i.e., the second oligonucleotide or the fourth
oligonucleotide, respectively.

[0050]FIG. 8A: depicts an electropherogram of exemplary ligated products
(indicated by arrow) generated using various first adaptors, various
second adaptors, or combinations of various first adaptors and various
second adaptors, as described in Example 5 and depicted in FIG. 8B.

[0051] X-axis:

[0052] T3r2-6 indicates that the corresponding ligation products were
generated in a ligation reaction with only first adaptors comprising
T3r2:27 6N (see FIG. 8B), where 6N equals six degenerate nucleotides and
r2 designates two 3' ribonucleotides;

[0053] T7-6 indicates that the corresponding ligation products were
generated in a ligation reaction with only second adaptors comprising
T7:6 N 28 (see FIG. 8B), where 6N equals six degenerate nucleotides;

[0054] T3r2-6+T7-6 indicates that the corresponding ligation products were
generated in a ligation reaction with first adaptors comprising T3r2:27
6N and second adaptors comprising T7: 6N 28, where and r2 designates two
3' ribonucleotides and where 6N equals six degenerate nucleotides in all
species of both adaptors;

[0055] rT3-6 indicates that the corresponding ligation products were
generated in a ligation reaction with only first adaptors comprising
rT3:27 6N, where 6N equals six degenerate nucleotides and rT3 designates
all ribonucleotides,

[0056] rT7-6 indicates that the corresponding ligation products were
generated in a ligation reaction with only second adaptors comprising
rT7:6 N 28, where rT7 designates all ribonucleotides, 6N equals six
degenerate nucleotides; and

[0057] rT3-6+rT7-6 indicates that the corresponding ligated products were
generated in a ligation reaction with first adaptors comprising rT3:27 6N
and second adaptors comprising rT7:6 N 28, where rT3 and rT7 designate
all ribonucleotides and 6N equals six degenerate nucleotides in all
species of both adaptors.

[0058]FIG. 8B schematically depicts two exemplary sets of first and
second adaptors of the current teachings (the two sets comprise
(i)rT3:27 6N (top first adaptor) and rT7:6 N 28 (top second adaptor)
and (ii) T3r2:27 6N (bottom first adaptor) and T7:6 N 28 (bottom second
adaptor), where 6N represents a series of six degenerate nucleosides on
the lower strand of the exemplary first adaptors and the lower strand of
the exemplary second adaptors.

[0059]FIG. 9A-FIG. 9c. FIG. 9A and FIG. 9B depict electropherograms of
exemplary ligated products generated according to certain embodiments of
the current teachings as described in Example 6. Three different
combinations of first adaptors and second adaptors were tested for double
ligation efficiency. These combinations included first adaptors and
second adaptors with both DNA upper strands (i.e., first and third
oligonucleotides) except for two ribonucleosides on the 3' end of the
first oligonucleotide, both RNA upper strands (i.e., first and third
oligonucleotides), or RNA upper strand on 5' (first) adaptor (i.e., first
oligonucleotide) and DNA upper strand on 3' (second) adaptor (i.e., third
oligonucleotide). FIG. 9c provides a schematic of the latter adaptor
structure embodiment having exemplary first adaptors (rT3: 27 6N) and
second adaptors (T7:6 N 28) (individually also described in FIG. 8B).

[0060] FIG. 10A and FIG. 10B depict two electropherograms showing
exemplary ligation products generated according to certain embodiments of
the current teachings using a series of ligation reaction compositions,
each comprising (1) Rnl2 ligase, (ii) a pool of synthetic miRNA molecules
(mirVana miRNA Reference Panel v 9.1, P/N 4388891 (Ambion, Austin, Tex.;
described herein) at a concentration of 2 and 0.2 picomoles (pmol), and
(iii) first and second adaptors as disclosed herein at upper to lower
strand ratios of 10/50, 5/25, 1/5, 1/50, 5/50, 10/50, 25/50, 5/100 or
5/500 (upper strand/lower strand) as shown and described in Example 6.

[0061]FIG. 11 schematically depicts certain embodiments of the current
teachings wherein various subpopulations of nucleic acid are removed
and/or purified from the sample. Embodiments of the methods can be used
for small RNA detection and isolation and for whole transcriptome
sequencing.

[0062]FIG. 12 depicts a graph of log2 fold change (FC) in
-ΔΔCT determined using an exemplary TaqMan®-based
detection of illustrative amplified products generated according to one
method of the current teachings (shown on y-axis as Log 2 (FC) TaqMan
(-ddCt)) versus the Log 2 (FC) determined using an exemplary sequencing
detection technique using the SOLiD® Sequencing System with an aliquot
of the same illustrative amplified products (shown on x-axis as Log 2 (FC
SOLiD®) as provided by Example 7.

[0063] FIG. 13A and FIG. 13B depict electropherograms comprising exemplary
amplified products generated by certain embodiments of the current
teachings visualized using SYBR® Gold staining as described in
Example 7.

[0064]FIG. 14 schematically depicts various embodiments of the current
teachings comprising detecting RNA molecules of interest by quantitating
exemplary amplified products generated according to the current teachings
using an intercalating dye, SYBR® Green (e.g., Example 8), for
detection in a real-time PCR reaction or SYBR® Gold staining of
electrophoretically separated amplified products ("SYBR® Assay").
LEGenD: Ligase Enhanced Gene Detection refers to use of double-strand
dependent ligase for assays as provided herein.

[0065]FIG. 15 schematically depicts an amplified product of the current
teachings as described in Example 8. P1 refers to a portion of the
forward PCR primer. P2 refers to a portion of the reverse PCR primer.

[0067]FIG. 17 provides an overview of a SOLiD® Small RNA Expression
Kit procedure for generating a small RNA library as provided in Example
11. Size-selected amplified small RNA enters the SOLiD® emulsion PCR
procedure at the "templated bead preparation" stage.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0068] It is to be understood that both the foregoing general description
and the following detailed description are exemplary and explanatory only
and are not intended to limit the scope of the current teachings. In this
application, the use of the singular includes the plural unless
specifically stated otherwise. For example, "a forward primer" means that
more than one forward primer can be present; for example, one or more
copies of a particular forward primer species, as well as one or more
different forward primer species. Also, the use of "comprise", "contain",
and "include", or modifications of those root words, for example but not
limited to, "comprises", "contained", and "including", are not intended
to be limiting. The term "and/or" means that the terms before and after
can be taken together or separately. For illustration purposes, but not
as a limitation, "X and/or Y" can mean "X" or "Y" or "X and Y".

[0069] The section headings used herein are for organizational purposes
only and are not to be construed as limiting the described subject matter
in any way. All literature and similar materials cited in this
application, including patents, patent applications, articles, books, and
treatises are expressly incorporated by reference in their entirety for
any purpose. In the event that one or more of the incorporated literature
and similar materials defines or uses a term in such a way that it
contradicts that term's definition in this specification, this
specification controls. While the present teachings are described in
conjunction with various embodiments, it is not intended that the present
teachings be limited to such embodiments. On the contrary, the present
teachings encompass various alternatives, modifications, and equivalents,
as will be appreciated by those of skill in the art.

[0070] The term "or combinations thereof" as used herein refers to all
permutations and combinations of the listed items preceding the term. For
example, "A, B, C, or combinations thereof" is intended to include at
least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a
particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB.
Continuing with this example, expressly included are combinations that
contain repeats of one or more item or term, such as BB, AAA, AAB, BBC,
AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will
understand that typically there is no limit on the number of items or
terms in any combination, unless otherwise apparent from the context.

[0071] According to certain disclosed methods, for example but not limited
to, the exemplary embodiment shown in FIG. 1, a ligation reaction
composition is formed comprising at least one RNA molecule to be
detected, at least one first adaptor, at least one second adaptor, and a
double-strand specific RNA ligase (shown as Hybridization of First and
Second Adaptors to RNA Molecule). Typically, the starting material
comprises a multiplicity of RNA species and multiplicities of different
first adaptors and different second adaptors.

[0072] As shown in FIG. 2A and FIG. 2B, the at least one first adaptor 21
comprises a first oligonucleotide 21A comprising at least two
ribonucleosides on the 3'-end and a second oligonucleotide 21B that
comprises a single-stranded 5' portion 21C when the first oligonucleotide
21A and the second oligonucleotide 21B are hybridized together (FIG. 2A),
and wherein the at least one second adaptor 22 comprises a third
oligonucleotide 22A that comprises a 5' phosphate group (shown as "P" in
FIG. 2B) and a fourth oligonucleotide 22B that comprises a
single-stranded 3' portion 22C when the third oligonucleotide 22A and the
fourth oligonucleotide 22B are hybridized together (FIG. 2A). It is to be
appreciated that in this illustrative embodiment, all of the nucleosides
in the first oligonucleotide are ribonucleosides, but that in other
embodiments as many as all and as few as two of the nucleosides of the
first oligonucleotide can be ribonucleosides, provided that the two
3'-most nucleosides of the first oligonucleotide 21A are ribonucleosides;
the remainder of the first oligonucleotide may comprise ribonucleosides,
deoxyribonucleosides, or a combination of both.

[0073] The single-stranded portions of the illustrative second and fourth
oligonucleotides (21C of 21B and 22C of 22B, respectively) of FIG. 2A are
depicted as degenerate hexamer sequences (shown as NNNNNN). However, the
use of first and/or second adaptors with sequence-specific single
stranded portions and also longer and shorter single-stranded portions is
within the scope of the current teachings. In some embodiments, the
degenerate sequences are deoxyribonucleotides. In some embodiments, the
length of the degenerate sequences is 4, 6, or 8 nucleotides. In some
embodiments, the first oligonucleotide comprises ribonucleosides and the
second, third and fourth oligonucleotides comprise deoxyribonucleotides.

[0074] First and second oligonucleotides are designed to be substantially
complementary with the exception of the single stranded portion 21C. The
substantially complementary portion can have a length of 10 to 60
nucleotides. When annealed or duplexed to form a first adaptor, the first
and second oligonucleotides can have one blunt end (as in FIG. 2A and
FIG. 2B) or can have an overhang of 1, 2, or 3 nucleotides at the end
opposite the end having a single stranded portion. The overhang may be on
either the first or the second oligonucleotide.

[0075] Third and fourth oligonucleotides are designed to be substantially
complementary with the exception of the single stranded portion 22C. The
substantially complementary portion can have a length of 10 to 60
nucleotides. When annealed or duplexed to form a second adaptor, the
third and fourth oligonucleotides can have one blunt end (as in FIG. 2A
and FIG. 2B) or can have an overhang of 1, 2, or 3 nucleotides at the end
opposite the end having a single stranded portion. The overhang may be on
either the first or the second oligonucleotide.

[0076] Returning to FIG. 1, the ligation reaction composition is incubated
under conditions suitable for a first adaptor and a second adaptor to
anneal with an RNA molecule. The first and third oligonucleotides ("upper
strands") may be present in a 1:1 to 1:10 molar ratio to the second and
fourth oligonucleotides ("lower strands"). In some embodiments, the molar
ratio of "upper strands" to "lower strands" is 1:5 or 1:2. A polypeptide
comprising double-strand specific RNA ligase activity is used to ligate
the annealed first adaptor-RNA molecule-second adaptor complex to form a
ligated product (shown as "Ligation" in FIG. 1). The first adaptor and
the second adaptor are ligated to the RNA molecule in the same reaction
composition, rather than as two separate sequential ligation reactions
with one or more intervening separation or purification steps between
ligating the two adaptors to the RNA molecule. It is to be understood the
order in which the components of the ligation reaction composition are
added and the sequence in which the two adaptors are ligated to the RNA
molecule are typically not limitations of the current teachings provided.

[0077] As shown in FIG. 2B, in certain embodiments, the first and second
adaptors are hybridized to the RNA molecule such that (i) the 3' end of
the first oligonucleotide of a first adaptor and the 5' end of the RNA
molecule are adjacently annealed to form a first ligation junction (for
example, 24 in FIG. 2B) (due to complementarity between the RNA molecule
and the single stranded portion of the first adaptor) and (ii) the 5' end
of third oligonucleotide of a second adaptor and 3' end of the same RNA
molecule are adjacently annealed to form a second ligation junction (for
example, 25 in FIG. 2B) (due to complementarity between the RNA molecule
and the single stranded portion of the second adaptor), wherein the first
and the second ligation junctions are both suitable for ligation using a
polypeptide comprising double strand-specific RNA ligase activity. In
some embodiments, the polypeptide comprising double strand-specific RNA
ligase activity comprises an Rnl2 family ligase, including without
limitation, Rnl2 ligase.

[0078] An RNA-directed DNA polymerase is combined with the ligated
product, along with suitable nucleotide triphosphates and a buffer
solution comprising appropriate salts. This reaction mixture is incubated
under conditions suitable for a reverse transcribed product to be
generated using the ligated product as the template (shown as "Reverse
Transcription" in FIG. 1). A separate reverse transcription primer is not
needed since the fourth oligonucleotide serves as the RT primer.

[0079] In some embodiments, the reverse transcribed product is placed on
an array and detected using standard methods known by one of skill in the
art. In some embodiments, the reverse transcribed product is labeled with
biotin and detection is by using streptavidin binding thereto. In some
embodiments, the reverse transcribed product is purified using glass
fiber filters, beads or is gel-purified. In some embodiments, the reverse
transcribed product is combined with a peptide comprising ribonuclease
activity to form a digestion reaction composition and incubated under
conditions suitable for digesting at least some of the ribonucleosides
from the reverse transcribed product to form an amplification template.
In some embodiments, the peptide comprising ribonuclease activity
comprises ribonuclease H(RNase H) activity (shown as "RNase H Digestion"
in FIG. 1).

[0080] The amplification template is combined with at least one forward
primer, at least one reverse primer, and a peptide comprising
DNA-directed DNA polymerase activity to form an amplification reaction
composition. When a DNA polymerase having both RNA-directed and
DNA-directed polymerase activities is used in the reverse transcription
reaction above, a further peptide comprising DNA-directed DNA polymerase
does not need to be added. The amplification reaction composition is
thermocycled under conditions suitable to allow amplified products to be
generated (shown as "Amplification" in FIG. 1). The sequence of at least
part of the amplified product is determined, which allows the
corresponding RNA molecule to be detected (shown as "Sequence
Determination" in FIG. 1).

[0081] According to one exemplary embodiment, depicted schematically in
FIG. 3, a population of small RNA molecules 33 is combined with a first
adaptor 31 comprising a first oligonucleotide comprising RNA (shown in
the open box) and a second adaptor 32 and Rnl2 ligase to form a ligation
reaction composition. The ligation reaction composition is incubated
under conditions suitable for annealing to occur and a first adaptor and
a second adaptor anneal with a small RNA molecule to form a ligation
template comprising the first adaptor annealed to the 5'-end of the RNA
molecule and the second adaptor annealed to the 3'-end of the small RNA
molecule. The ligase will generate a ligated product 34 by ligating the
first adaptor to the 5'-end of the RNA molecule and the second adaptor to
the 3'-end of the RNA molecule at the ligation junctions (shown as solid
dots in FIG. 3 and indicated by arrows). Depending on the concentration
of adaptors and RNA molecules in the ligation reaction composition, some
unannealed first adaptors 31 and/or second adaptors 32 may also be
present in the ligation reaction composition. Additionally, particularly
when the first and/or second adaptors comprise degenerate sequences,
undesired annealed byproduct molecules 35 can also form.

[0082] The reaction composition comprising the ligated product is combined
with an RNA-directed DNA polymerase and under suitable conditions a
reverse transcribed product 36 is generated. The reaction composition
comprising the reverse transcribed product 36 is combined with
ribonuclease H and at least some of the ribonucleosides of the ligated
product are digested and an amplification template 38 is generated. Those
in the art will appreciate that, at this point, the amplification
template comprises, in essence, the cDNA strand of the reverse
transcribed product annealed with the third oligonucleotide of the second
adaptor. The amplification template 38 is combined with a DNA-directed
DNA polymerase, a forward primer 310 and a reverse primer 311 to form an
amplification reaction composition. In this illustrative embodiment, the
reverse primer further comprises an identification sequence 312,
sometimes referred to as a "bar code" sequence. If all of the reverse
primers in a given amplification reaction composition comprise the same
identification sequence and that sequence is incorporated into subsequent
amplicons, then all of the amplicons generated from the same
amplification reaction composition can be identified as having come from
that reaction composition.

[0083] The amplification reaction composition is temperature cycled to
allow the polymerase chain reaction to occur and a plurality of amplified
products is generated. In this illustrative embodiment, the amplified
products are purified using polyacrylamide gel electrophoresis (PAGE)
and/or high performance liquid chromatography (HPLC; sometimes referred
to as high pressure liquid chromatography). The purified amplified
products are sequenced using any technique known in the art, and the RNA
molecule corresponding to that sequence is identified. Those in the art
will appreciate that the disclosed method may be useful for a variety of
analyses, including without limitation, expression profiling,
quantitating one or more specific RNA molecules in one or more
corresponding samples (e.g., with and without drug treatment; a
malignant/tumor tissue and the corresponding normal tissue sample;
developmental studies using corresponding embryonic, neonatal,
adolescent, and/or adult tissues), and small RNA discovery.

[0084] It is to be appreciated that if multiple libraries of amplified
product are to be generated, each amplified product library can be
identified by a unique identification sequence or barcode for that
library. In some embodiments, the PCR primer mix for a given amplified
product library comprises a forward primer (for illustration purposes,
see forward primer 310 in FIG. 3) and a reverse primer that contains a
unique identification sequence or barcode (for illustration purposes, see
reverse primer 311 comprising identifier sequence 312 in FIG. 3). When an
amplification reaction composition comprising such a primer pair is
cycled, the barcode is becomes incorporated into the amplified products
of that library. Thus, the exemplary first primer can be matched with any
of these exemplary reverse primers in an amplification reaction
composition to generate a library of amplified products comprising the
barcode of the reverse primer or its complement.

[0085] For illustration purposes but not as a limitation, each amplified
product in a first library generated using a PCR primer mix including the
exemplary forward primer and exemplary reverse primer BC1 (see Example
11) will contain the barcode sequence AAGCCC and/or its complement; while
each amplified product in a second library generated using a PCR primer
mix including the exemplary forward primer and exemplary reverse primer
BC2 will contain the barcode sequence CACACC and/or its complement; and
so forth. Thus, multiple libraries of amplified product can be pooled
prior to sequencing and the RNA molecules in the starting material
corresponding to each library can be identified using the target (RNA
molecule) sequence or at least part of that sequence combined with the
barcode or identification sequence for that library. Those in the art
will appreciate that various identification sequences can be employed to
uniquely mark the amplified products generated in a given amplification
reaction composition.

[0086]FIG. 4 schematically depicts another exemplary embodiment of the
current teachings. According to this embodiment, the first and second
adaptors are hybridized with the RNA molecule (shown as Adaptor
Hybridization in FIG. 4) and under suitable conditions and in the
presence of an appropriate ligase, ligated product is generated (shown as
Ligation in FIG. 4). Reverse transcriptase is added to the ligated
product and a reverse transcribed product is generated under suitable
conditions (shown as Reverse Transcription in FIG. 4). The reverse
transcribed product is digested with ribonuclease H and an amplification
template is formed (shown as RNase H in FIG. 4). An amplification
reaction composition is formed comprising the amplification template,
forward primer, reverse primer, and a DNA-directed DNA polymerase. The
amplification reaction composition is thermocycled for a number of cycles
that stays within a linear range of amplification (generally,
˜12-15 cycles or 12-18 cycles according to one exemplary
embodiment), allowing the polymerase chain reaction to occur and
amplified product to be generated (shown as PCR in FIG. 4). In this
illustrative embodiment, the amplified product is gel purified (shown as
Gel Purif. in FIG. 4) resulting in purified amplified product. Provided
that appropriately size fractionated or fragmented RNA molecules were
used, the amplified product comprises an insert sequence (shown by a
curved bracket in FIG. 4; in certain embodiments, insert sizes are about
15 base pairs to about 100 base pairs), a first primer region (shown as
P1 in FIG. 4), and a second primer region (shown as P2 in FIG. 4) that
includes a bar code or identification sequence (shown as be in FIG. 4).

[0087] As used herein, the terms "polynucleotide", "oligonucleotide", and
"nucleic acid" are used interchangeably and refer to single-stranded and
double-stranded polymers of nucleoside monomers, including
2'-deoxyribonucleosides (DNA) and ribonucleosides (RNA) linked by
internucleotide phosphodiester bond linkages, or internucleotide analogs,
and associated counter ions, e.g., H.sup.+, NH4.sup.+,
trialkylammonium, Mg2+, Na.sup.+, and the like. A polynucleotide may
be composed entirely of deoxyribonucleosides, entirely of
ribonucleosides, or chimeric mixtures thereof. As further described
below, for example, first adaptors include a first oligonucleotide having
at least two ribonucleosides on its 3' end. First oligonucleotides can
have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more
ribonucleosides and in some embodiments, the ribonucleosides are
contiguous. In some embodiments, second, third or fourth oligonucleotides
comprise deoxyribonucleotides. The nucleotide monomer units may comprise
any of the nucleotides described herein, including, but not limited to,
nucleotides and nucleotide analogs. Polynucleotides typically range in
size from a few monomeric units, e.g. 5-40 or 5-60 when they are
sometimes referred to in the art as oligonucleotides, to several
thousands of monomeric nucleotide units. Unless denoted otherwise,
whenever a polynucleotide sequence is represented, it will be understood
that the nucleotides are in 5' to 3' order from left to right.

[0088] RNA Molecule to be Detected:

[0089] In some embodiments, the RNA molecules of the current teachings
comprise total RNA, a subset or fraction of total RNA, or both. In some
embodiments, a sample comprising the RNA molecule to be detected
comprises all of the RNA obtained from a particular sample or pool of
samples. In other embodiments, the total RNA is fractionated into subsets
and the RNA molecule to be detected is present in one or more of the
fractionated subsets. Typically, RNA molecules are extracted from a
sample using any technique known in the art that yields total RNA or a
subset of RNA molecules in the sample.

[0090] In some embodiments, the RNA molecules to be detected are
fragmented, typically prior to forming the ligation reaction composition.
In some embodiments, total RNA can be fragmented or fractionated RNA can
be fragmented and analyzed using methods provided herein. In some
embodiments, the RNA molecule to be detected comprises a plurality of
different RNA species, including without limitation, a plurality of
different mRNA species, which may or may not be fragmented prior to
generating ligation products. In some embodiments the RNA is fragmented
chemically, enzymatically, mechanically, by heating, or combinations
thereof using methods well known in the art. Fragmented RNA is analyzed
using methods provided herein. Whole transcriptome analysis can thus be
carried out wherein sequences that are transcribed from DNA are analyzed,
including coding RNA (e.g., for expression analysis) or noncoding RNA.

[0091] In some embodiments, small RNA molecules and/or fragmented RNA in a
certain size range are obtained from a sample, for example using a size
fractionation procedure. In some embodiments, the total RNA is fragmented
and may also be size fractionated prior to ligating the first and second
adaptors; while in other embodiments total RNA is used in the ligation
reaction composition. For illustration purposes, but not as a limitation,
certain fractionation techniques are depicted in FIG. 11.

[0092] In some embodiments, a poly A selection process is performed to
separate messenger RNA (mRNA) from those RNA molecules that lack poly A
(poly A minus RNA molecules). In some embodiments, the total RNA is
fractionated into subsets by separating at least some of the mRNA from
the total RNA, for example but not limited to, using a polyA selection
technique known in the art, including without limitation, oligo-dT
chromatography. In such embodiments, either the poly A+ fraction or the
poly A depleted fraction may be employed in the current teachings to
detect at least some of the RNA molecules that are present in that
fraction. In some embodiments, both fractions may be used separately to
detect at least some of the RNA molecules that are present in each
fraction. In some embodiments, the RNA molecules of interest comprise
poly A minus RNA molecules, for example but not limited to large
non-coding RNA.

[0093] In certain embodiments, a population of mRNA molecules or a
population of poly A minus RNA molecules is depleted of at least one
species of abundant RNA molecule in the population, for example but not
limited to, ribosomal RNA, or mRNA from housekeeping or highly expressed
genes, including without limitation, actin mRNA and globin mRNA. For
example, certain mRNAs or classes of RNA are depleted from the total RNA,
for example but not limited to, high copy number mRNAs such as actin,
GAPDH, globin and other "housekeeping" mRNA; and classes of RNA for
example but not limited to 18S RNA and 28S RNA (for example, using
commercially available kits such as the RiboMinus (Invitrogen, Carlsbad,
Calif.) or GLOBINcIear® (Ambion, Austin, Tex.) Kits (see also U.S.
Patent Application Publication US 2006/0257902, Methods and Compositions
for Depleting Abundant RNA Transcripts).

[0094] The term chemical fragmentation is used in a broad sense herein and
includes without limitation, exposing the sample comprising the RNA to
metal ions, for example but not limited to, zinc (Zn2+), magnesium
(Mg2+), and manganese (Mn2+) and heat.

[0095] The term enzymatic fragmentation is used in a broad sense and
includes combining the sample comprising the RNA with a peptide
comprising nuclease activity, such as an endoribonuclease or an
exoribonuclease, under conditions suitable for the peptide to cleave or
digest at least some of the RNA molecules. Exemplary nucleases include
without limitation, ribonucleases (RNases) such as RNase A, RNase T1,
RNase T2, RNase U2, RNase PhyM, RNase III, RNase PH, ribonuclease V1,
oligoribonuclease (e.g., EC 3.1.13.3), exoribonuclease I (e.g., EC
3.1.11.1), and exoribonuclease II (e.g., EC 3.1.13.1), however any
peptide that catalyzes the hydrolysis of an RNA molecule into one or more
smaller constituent components is within the contemplation of the current
teachings. Fragmentation of RNA molecules by nucleic acids, for example
but not limited to, ribozymes, is also within the scope of the current
teachings.

[0096] The term mechanical fragmentation is used in a broad sense and
includes any method by which nucleic acids are fragmented upon exposure
to a mechanical force, including without limitation, sonication,
collision or physical impact, and shear forces.

[0097] In some embodiments, very small fragments of RNA are removed using
a "clean up" step, for example but not limited to, purification using gel
electrophoresis, glass fiber filters or using magnetic beads, prior to
using the remaining larger RNA molecules according to the current
teachings.

[0098] In certain embodiments, the methods of the current teachings employ
RNA molecules that were fractionated using a physical separation method,
including without limitation, size separation methods such as
centrifugation, column chromatography/gel sieving, and electrophoretic
separation. In some embodiments, electrophoretic separation of RNA
molecules of interest comprise the flashPAGE® Fractionator System
(Ambion, Austin, Tex.) or size selection by slicing a band from an
agarose or polyacrylamide gel according to methods known in the art. In
some embodiments, the RNA molecules used in certain disclosed methods can
be obtained by extracting a subset of RNA molecules in a sample using any
of a variety of sample preparation kits and reagents, including without
limitation, the mirVana® miRNA Isolation Kit (Ambion). In some
embodiments, RNA may be immunoprecipitated.

[0100] Those of skill in the art will appreciate that the length of RNA
molecule to be detected is not a limitation of the current teachings
since longer molecules can be fractionated and/or fragmented and the
fractions and/or fragments detected so as to reconstruct the RNA
molecule. In some embodiments, the length of the RNA molecule to be
detected is 12 to 500 nucleotides, 15 to 110 nucleotides, 15 to 100
nucleotides, 18 to 110 nucleotides, 20 to 80 nucleotides, 25 to about 60
nucleotides, 20 to about 45 nucleotides, 20 to about 41 nucleotides, 20
to about 40 nucleotides, 21, 22, 23, 24, or 25 to about 36, 37, 38, 39,
40, or 41 nucleotides, or any integer range therebetween. In some
embodiments, the RNA molecule to be detected has a length of 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, or 41 nucleotides.

[0101] Those in the art will appreciate that the techniques used to
fractionate or fragment RNA is not a limitation of the current teachings
and that various fractionation or fragmentation techniques can typically
be employed, depending on which fraction(s) or fragment(s) of RNA
molecules is to be detected.

[0102] In certain embodiments, the starting material comprises at least
one synthetic RNA molecule, such as a spike-in control that may be used
for, among other things, calibration or standardization. In some
embodiments, at least one synthetic RNA molecule species is added to a
sample comprising naturally occurring RNA molecules and the presence of
at least one synthetic RNA species and at least one naturally occurring
RNA species is detected according to the disclosed methods.

[0103] In some embodiments, the RNA molecule to be detected has a
5'-monophosphate and a 3'-hydroxyl for efficient ligation. For example,
some small RNA biogenesis results in RNA molecules with 5'-ends
comprising triphosphates. According to certain embodiments of the current
teachings, such RNA molecules are not suitable for adaptor ligation and
amplification. Thus, intact mRNA molecules with a 5' cap structure, and
RNA molecules with a 5' triphosphate, including small RNAs such as
endogenous siRNA from C. elegans (Pak 2007), cannot be effectively
ligated to the hybridized adaptors in the reaction composition, unless
they are first treated with a decapping enzyme, for example but not
limited to, tobacco acid pyrophosphatase (TAP), nuclease P1, Dcp1p
decapping enzyme, Dcp2 decapping enzyme, or DcpS decapping enzyme to
convert the 5' ends of RNA molecules to 5' monophosphates. Where the RNA
molecule of interest comprises 5'-triphosphates, certain embodiments of
the current teachings employ tobacco acid pyrophosphatase to convert the
5'-ends of RNA molecules to 5' monophosphates, rendering them suitable
for ligation.

[0104] In some embodiments, fragments generated by certain fragmentation
techniques do not initially possess a terminus that is suitable for
enzymatic ligation; in some embodiments, such fragments are treated with
a kinase, for example but not limited to, bacteriophage T4 polynucleotide
kinase, to render the 5'-ends or 3'-ends suitable for ligating according
to the current teachings.

[0105] RNA to be detected may be single stranded or double stranded since
the RNA to be detected is combined with at least one first adaptor, at
least one second adaptor, and annealed such that a polypeptide comprising
double-strand specific RNA ligase can form a ligated product.

[0106] According to the current teachings, an RNA molecule of interest can
be either synthetic or naturally occurring. RNA molecules can be
synthesized using oligonucleotide synthesis methods that are well-known
in the art. RNA molecules can also be synthesized biochemically, in vivo
or in vitro, according to methods known in the art, for example but not
limited to in vitro transcription techniques, including without
limitation, U.S. Pat. Nos. 5,958,688; 5,723,290; 5,514,545; 5,021,335;
5,168,038; 5,545,522; 5,716,785; 5,891,636; and 6,291,170. Detailed
descriptions of such techniques can be found in, among other places,
Current Protocols in Nucleic Acid Chemistry, Beaucage et al., eds., John
Wiley & Sons, New York, N.Y., including updates through May 2005
(hereinafter "Beaucage et al."); and Blackburn and Gait. Automated
nucleic acid synthesizers useful for synthesizing RNA molecules,
adaptors, and primers are commercially available from numerous sources,
including for example, Applied Biosystems (Foster City, Calif.). RNA
molecules, adaptors, and primers can also be generated biosynthetically,
using in vivo methodologies and/or in vitro methodologies that are well
known in the art. Descriptions of such technologies can be found in,
among other places, Sambrook et al. and Ausubel et al. Nucleoside
analogs, such as 2'-OMe-, LNA-, halo-, or arabino-derivatives, for
example, or universal nucleobases can be incorporated into adaptors as
long as the fourth oligonucleotide is a primeable substrate. Purified or
partially purified RNA is commercially available from numerous sources,
including FirstChoice® Total RNA, FirstChoice® Poly(A),
FirstChoice® Tumor RNA, and the mirVana® miRNA Reference Panel
(Ambion, Austin, Tex.); Reference Total RNA, Human and Mouse, and
Universal Reference RNAs (Stratagene, La Jolla, Calif.); and the American
Type Culture Collection (ATCC), Manassas, Va.

[0107] In some embodiments, the RNA molecule to be detected is present in
a sample. The term "sample" is used in a broad sense herein and is
intended to include a wide range of biological materials as well as
compositions derived or extracted from such biological materials
comprising or suspected of comprising RNA. Exemplary samples include
whole blood; red blood cells; white blood cells; buffy coat; hair; nails
and cuticle material; swabs, including buccal swabs, throat swabs,
vaginal swabs, urethral swabs, cervical swabs, rectal swabs, lesion
swabs, abcess swabs, nasopharyngeal swabs, and the like; urine; sputum;
saliva; semen; lymphatic fluid; amniotic fluid; cerebrospinal fluid;
peritoneal effusions; pleural effusions; fluid from cysts; synovial
fluid; vitreous humor; aqueous humor; bursa fluid; eye washes; eye
aspirates; plasma; pulmonary lavages; lung aspirates; and tissues,
including, liver, spleen, kidney, lung, intestine, brain, heart, muscle,
pancreas, biopsy material, and the like. The skilled artisan will
appreciate that lysates, extracts, or materials obtained from any of the
above exemplary biological samples are also within the scope of the
current teachings. Tissue culture cells, including explanted material,
primary cells, secondary cell lines, and the like, as well as lysates,
extracts, or materials obtained from any cells, are also within the
meaning of the term biological sample as used herein. Materials
comprising or suspected of comprising at least one RNA molecule that are
obtained from forensic, agricultural, and/or environmental settings are
also within the intended meaning of the term sample. In certain
embodiments, a sample comprises a synthetic nucleic acid sequence. In
some embodiments, a sample is totally synthetic, for example but not
limited to, a control sample comprising a buffer solution containing at
least one synthetic nucleic acid sequence. In certain embodiments, the
sample is an environmental sample, such as a soil, water, or air sample.

[0108] Plant miRNAs can have a 2'-O-methyl group at the 3' end and can be
ligated in a ligation reaction as cited herein. However, the efficiency
of such a ligation will be reduced compared to RNA species with a 2'-OH
at the 3' end.

[0109] First Adaptors and Second Adaptors:

[0110] As stated above, at least one first adaptor comprises a first
oligonucleotide comprising at least two ribonucleosides on the 3'-end and
a second oligonucleotide that comprises a single-stranded 5' portion when
the first oligonucleotide and the second oligonucleotide are hybridized
together as depicted in FIG. 2A. First and second oligonucleotides are
designed to be substantially complementary with the exception of the
single stranded portion, further described below. The substantially
complementary portion can have a length of 10 to 60 nucleotides. In some
embodiments, the substantially complementary portion can have a length of
10 to 40 nucleotides, 12 or 15 to 30 nucleotides, 20, 21, 22, or 23 to
25, 27 or 29 nucleotides, or any integer range between any of these
ranges. When annealed or duplexed to form a first adaptor, the first and
second oligonucleotides can have one blunt end (as in FIG. 2A and FIG.
2B) or can have an overhang of 1, 2, or 3 nucleotides at the end opposite
the end having a single stranded portion. The overhang may be on either
the first or the second oligonucleotide.

[0111] Also as stated above, at least one second adaptor comprises a third
oligonucleotide that comprises a 5' phosphate group and a fourth
oligonucleotide that comprises a single-stranded 3' portion when the
third oligonucleotide and the fourth oligonucleotide are hybridized
together also as depicted in FIG. 2A. Third and fourth oligonucleotides
are designed to be substantially complementary with the exception of a
single stranded portion, further described below. The substantially
complementary portion can have a length of 10 to 60 nucleotides. When
annealed or duplexed to form a second adaptor, the third and fourth
oligonucleotides can have one blunt end (as in FIG. 2A and FIG. 2B) or
can have an overhang of 1, 2, or 3 nucleotides at the end opposite the
end having a single stranded portion. The overhang may be on either the
first or the second oligonucleotide.

[0112] In some embodiments, first, second, third and fourth
oligonucleotides independently comprise deoxyribonucleosides,
ribonucleotides, or both deoxyribonucleotides and ribonucleotides with
the exception that the first oligonucleotide comprises at least two
ribonucleosides on the 3'-end. It is to be appreciated that in the
illustrative embodiment of FIG. 2A, all of the nucleosides in the first
oligonucleotide are ribonucleosides, but that in other embodiments as
many as all and as few as two of the nucleosides of the first
oligonucleotide can be ribonucleosides, provided that the two 3'-most
nucleosides of the first oligonucleotide are ribonucleosides; the
remainder of the first oligonucleotide may comprise ribonucleosides,
deoxyribonucleosides, or a combination of both. In some embodiments, the
second, third and fourth oligonucleotides comprise deoxyribonucleosides
and the first oligonucleotide comprises all ribonucleosides. In some
embodiments, the first oligonucleotide comprises ribonucleosides and the
second, third and fourth oligonucleotides comprise deoxyribonucleotides.
The length of oligonucleotides of first and second adaptors is
independent of each other.

[0113] The sequences of the first, second, third and fourth
oligonucleotides are such that substantial complementarity is achieved in
the duplexed portion of the adaptors as described above. In some
embodiments, the sequences of the first and the third oligonucleotides
are different. The specific sequence of nucleotides of the duplexed
portions of the adaptors is not limiting for the methods herein. In some
embodiments, a portion of an adaptor sequence comprises a "promoter
sequence," including without limitation a sequence suitable for
initiating transcription using a suitable polymerase, for example but not
limited to, T3 RNA polymerase, T7 RNA polymerase, or SP6 RNA polymerase.
In some embodiments, a first adaptor comprises a "promoter sequence" for
a first promoter and a second adaptor comprises a "promoter sequence" for
a second promoter.

[0114] The 3'-end of the first oligonucleotide and the 5'-end of the third
oligonucleotide are suitable for ligation to an RNA molecule to be
detected, which also is suitable for ligation. Oligonucleotides "suitable
for ligation" refers to at least one RNA molecule to be detected, and at
least one first adaptor and/or at least one second adaptor, each
comprising an appropriate reactive group. Exemplary reactive groups
include, but are not limited to, a free hydroxyl group on the 3' end of
the first oligonucleotide of a first adaptor and a free phosphate group
on the 5' end of the RNA molecule to be detected, a free hydroxyl group
on the 3' end of the RNA molecule to be detected and a free phosphate
group on the 5' end of the third oligonucleotide of a second adaptor.

[0115] Single-Stranded Portions of Adaptors:

[0116] The single-stranded portions of the illustrative second and fourth
oligonucleotides are depicted in FIG. 2A as degenerate hexamer sequences
(shown as NNNNNN). However, the use of first and/or second adaptors with
sequence-specific single-stranded portions and also longer and shorter
single-stranded portions is within the scope of the current teachings.

[0117] In some embodiments, the single-stranded portions comprise,
independently, deoxyribonucleosides, ribonucleosides or a combination of
deoxyribonucleosides and ribonucleosides. In some embodiments, the
single-stranded portions comprise deoxyribonucleosides.

[0118] In some embodiments of single-stranded portions of adaptors, the
length of the single-stranded portion is as short as one nucleotide and
as long as 8 nucleotides. In some embodiments, the length of the
single-stranded portion is 2, 4, 6, or 8 nucleotides. In some
embodiments, the length of the single-stranded portion is 4 or 6
nucleotides. The length of the single-stranded portion of the second
oligonucleotide is independent of the length of the single-stranded
portion of the fourth oligonucleotide.

[0119] In some embodiments the nucleoside sequence of a single-stranded
portion is designed to be complementary to a 5'-sequence or a 3'-sequence
of a specific RNA molecule to be detected. In some embodiments, the
specific RNA molecule to be detected hybridizes with the single-stranded
portion of at least one first adaptor and the single-stranded portion of
at least one second adaptor such that a ligation reaction can occur. For
hybridizing to specific sequences in some embodiments, the length of the
single-stranded portions are independently 4 to 6 nucleotides long. In
such a method, the RNA molecule is directionally detected by methods
herein. One of ordinary skill in the art can design a single-stranded
portion corresponding to the 5'-sequence or a 3'-sequence of the RNA
molecule to be detected and using the detection methods provided herein
detect either the sense sequence or the antisense sequence corresponding
to the RNA molecule.

[0120] In some embodiments, the sequence of a single-stranded portion is
designed to be a degenerate sequence to allow all RNA molecules of a
sample having complementary to the degenerate sequence to anneal to the
single-stranded portion of the adaptor. In some embodiments, degenerate
single-stranded portions have a length of 1 to 8 nucleotides. In some
embodiments, degenerate single-stranded portions have a length of 4, 6,
or 8 nucleotides. In some embodiments, the degenerate nucleoside
sequences are deoxyribonucleotides.

[0121] In some embodiments, the sequence of a single-stranded portion of a
second or fourth oligonucleotide is a degenerate sequence and the
sequence of the other of the second or fourth oligonucleotide is a
sequence corresponding to the RNA molecule to be detected.

[0122] Annealing or Hybridizing:

[0123] The terms "annealing" and "hybridizing" including, without
limitation, variations of the root words hybridize and anneal, are used
interchangeably and mean the nucleotide base-pairing interaction of one
nucleic acid with another nucleic acid that results in the formation of a
duplex, triplex, or other higher-ordered structure. The primary
interaction is typically nucleotide base specific, e.g., A:T, A:U, and
G:C, by Watson-Crick and Hoogsteen-type hydrogen bonding. In certain
embodiments, base-stacking and hydrophobic interactions may also
contribute to duplex stability. For example, conditions under which
primers anneal to complementary or substantially complementary sequences
are well known in the art, e.g., as described in Nucleic Acid
Hybridization, A Practical Approach, Hames and Higgins, eds., IRL Press,
Washington, D.C. (1985) and Wetmur and Davidson, Mol. Biol. 31:349, 1968.
In general, whether annealing takes place is influenced by, among other
things, the length of the complementary portion of the nucleic acids, the
pH, the temperature, the presence of mono- and divalent cations, the
proportion of G and C nucleotides in the hybridizing region, the
viscosity of the medium, and the presence of denaturants. Such variables
influence the time required for hybridization. The presence of certain
nucleotide analogs or minor groove binders in the complementary portions
of nucleic acids can also influence hybridization conditions. Thus, the
preferred annealing conditions will depend upon the particular
application. Such conditions, however, can be routinely determined by
persons of ordinary skill in the art, without undue experimentation.
Typically, annealing conditions are selected to allow nucleic acids to
selectively hybridize with a complementary or substantially complementary
sequence, but not hybridize to any significant degree to other sequences
in the reaction.

[0124] The term "selectively hybridize" and variations thereof means that,
under suitable conditions, a given sequence anneals with a second
sequence comprising a complementary or a substantially complementary
string of nucleotides, but does not anneal to undesired sequences. In
this application, a statement that one sequence selectively hybridizes or
anneals with another sequence encompasses situations where the entirety
of both of the sequences hybridize to one another, and situations where
only a portion of one or both of the sequences hybridizes to the entire
other sequence or to a portion of the other sequence. For the purposes of
this definition, the term "sequence" includes nucleic acid sequences,
polynucleotides, oligonucleotides, primers, target-specific portions,
amplification product-specific portions, primer-binding sites,
hybridization tags, and hybridization tag complements.

[0125] The term "corresponding" as used herein refers to at least one
specific relationship between the elements to which the term relates. For
example, a single-stranded 5' portion of a first adaptor corresponds to a
RNA molecule having a terminal nucleotide sequence that hybridizes to the
single-stranded portion. A single-stranded 3' portion of a second adaptor
corresponds to a RNA molecule having a terminal nucleotide sequence that
hybridizes to the single-stranded portion. Further examples include where
a primer binds to the corresponding complementary or substantially
complementary primer-binding portion of a nucleic acid, where a
particular affinity tag binds to the corresponding affinity tag, for
example but not limited to, biotin binding to streptavidin, and where a
particular hybridization tag anneals with its corresponding hybridization
tag complement; and the like.

[0126] In this application, a statement that one sequence is the same as,
substantially the same as, complementary to, or substantially
complementary to another sequence encompasses situations where both of
the sequences are completely the same as, substantially the same as, or
complementary or substantially complementary to one another, and
situations where only a portion of one of the sequences is the same as,
substantially the same as, complementary to, or substantially
complementary to a portion or the entire other sequence. For the purposes
of this definition, the term "sequence" includes RNA, DNA,
polynucleotides, oligonucleotides, primers, ligated products, reverse
transcribed products, amplification templates, amplified products,
primer-binding sites, hybridization tags, and hybridization tag
complements.

[0127] The terms "denaturing" or "denaturation" as used herein refer to
any process in which a double-stranded polynucleotide, including a
double-stranded amplification product or a double-stranded DNA or a
DNA:RNA duplex is converted to two single-stranded polynucleotides.
Denaturing a double-stranded polynucleotide includes without limitation,
a variety of thermal or chemical techniques for denaturing a duplex,
thereby releasing its two single-stranded components. Those in the art
will appreciate that the denaturing technique employed is generally not
limiting unless it inhibits or appreciably interferes with a subsequent
amplifying and/or detection step.

[0128] Ligation:

[0129] The term "ligating," or forms thereof, is used herein to refer to
an enzymatic ligation process that uses a polypeptide comprising
double-strand specific RNA ligase activity in which an inter-nucleotide
linkage is formed between immediately adjacent ends of oligonucleotides
that are adjacently hybridized to a template. Formation of the linkage is
double-strand dependent and specific, also termed duplex-dependent and
specific or template-dependent and specific. The internucleotide linkage
can include, but is not limited to, phosphodiester bond formation between
a 3'-ribonucleoside and a 5'-ribonucleotide, or a 3'-ribonucleoside and a
5'-deoxyribonucleoside. The term "double-strand specific RNA ligase" as
used herein refers to a polypeptide comprising RNA ligase activity that
preferentially seals or ligates a nick between an oligonucleotide having
a 3'-terminal ribonucleotide and an oligonucleotide having a 5' phosphate
group, specifically when the oligonucleotides are immediately adjacently
hybridized to a template molecule. For example, but without limitation, a
nick between the 3'-end of the first oligonucleotide of the first adaptor
and the RNA molecule to which the first adaptor is annealed is
schematically presented as ligation junction 24 in FIG. 2B.

[0130] In certain embodiments, the polypeptide comprising double-strand
specific RNA ligase activity is an Rnl2 family ligase exemplified by the
bacteriophage T4 RNA ligase 2 (T4 Rnl2), including without limitation, an
enzymatically active mutant or variant of Rnl2. T4 Rnl2 is a prototype
ligase for an RNA ligase family that differs from the Rnl1 family of
ligases due to variant nucleotidyl transferase motifs (see, e.g., Ho and
Shuman, Proc. Natl. Acad. Sci. 99(20):12709-14 (2002); and Yin et al., J.
Biol. Chem. 278:17601-08 (2003)). The T4 Rnl2 family includes vibriophage
KVP40 Rnl2, the RNA-editing ligases (RELs) of Trypanosoma brucei (TbREL1
and TbREL2) and of Leishmania tarentolae (LtREL1 and LtREL2), poxvirus
AmEPV (entomopoxvirus) ligase, baculovirus AcNPV ligase, and baculovirus
XcGV ligase, among others. In some embodiments using REL ligases, the
second and fourth oligonucleotides can comprise ribonucleotides and, in
certain embodiments, the single stranded portions of the second and
fourth oligonucleotides can comprise ribonucleotides.

[0131] T4 Rnl2 ligase is commercially available from NEW ENGLAND
BIOLABS® (Ipswich, Mass.) or the ligase can be isolated as described
in Nandakumar et al., JBC 280(25):23484-23489, 2005; Nandakumar et al.,
JBC 279(30):31337-31347, 2004; and Nandakumar et al., Molecular Cell
16:211-221, 2004. The T4 Rnl2 enzyme is encoded by gene gp24.1 of phage
T4. In certain embodiments, a polypeptide comprising ligase activity
comprises T4 Rnl2 or another member of the Rnl2 family of ligases, or an
enzymatically active mutant or variant thereof.

[0132] In certain embodiments, the polypeptide comprising double-strand
specific RNA ligase activity is a Deinococcus radiodurans RNA ligase
(DraRnl) (Raymond et al., Nucl Acids Res 35(3):839-849, 2007), or a
DraRnl-type ligase, including without limitation, a ligase having Gen
Bank accession no. XP--367846 from the fungi Magnaporthe grisea, Gen
Bank accession no. CAE76396 from Neurospora crassa, accession no.
XP--380758 from Gibberella zeae, or Accession no. EAL61744 from the
amoeba Dictyostelium discoideum. In some embodiments, a ligase can
include a combination of any of the above-cited ligases, or enzymatically
active mutants or variants thereof.

[0133] In certain embodiments, a polypeptide comprising double-strand
specific RNA ligase activity can be preadenylated, the 5'-terminal
nucleotide of the third oligonucleotide can be preadenylated, or the
5'-terminal nucleotide of the RNA molecule to be detected can be
preadenylated, or a combination thereof. Ho et al. (Structure 12:327-339)
sets forth a mechanism for T4 Rnl2 where the C-terminal domain thereof
functions in sealing 3'-OH and 5'-P RNA ends. The N-terminal segment
(1-249) of the Rnl2 protein is reported to function as an autonomous
adenylyltransferase/App-RNA ligase domain. In general, RNA ligases join
3'-OH and 5'-PO4 RNA termini through a series of three nucleotidyl
transfer steps involving activated covalent intermediates. RNA ligase
reacts with ATP to form a covalent ligase-AMP intermediate plus
pyrophosphate. AMP is then transferred from ligase-adenylate to a
5'-PO4 RNA end to form an RNA-adenylate intermediate (AppRNA).
Ligase then catalyzes attack by an RNA 3'-OH on the RNA-adenylate to seal
the two ends via a phosphodiester bond and release AMP. Mechanisms for
RNA ligation are further discussed by Nandakumar et al. (ibid 2005,
2004a, 2004b) Yin et al. (JBC 278:20, 17601-17608; Virology 319:141-151,
2004), Ho et al. (ibid; PNAS, 99:20, 12709-12714, 2002), Gumport et al.
(in Gene Amplification and Analysis, Vol 2, edited by Chirikjian, J. G.,
and Papas, T. S., 1981, 313-345) and by Raymond et al. (Nucleic Acids
Res. 35:3, 839-849, 2007). Preadenylated agents such as ligase-adenylate,
RNA-adenylate, or a chimeric DNA/RNA-adenylate are contemplated for use
in some embodiments of the current teachings.

[0134] According to certain embodiments, at least one species of first
adaptor, at least one species of second adaptor, at least one species of
RNA molecule, and a polypeptide comprising double-strand specific RNA
ligase activity are combined in a ligation reaction composition. It is to
be appreciated that the adaptors of the current teachings are each
ligated with the corresponding RNA molecule in the same reaction
composition during the same incubation period, that is simultaneously or
nearly simultaneously, in contrast to other techniques in which one
adaptor is ligated to one end of an RNA molecule of interest in one
reaction, then another adaptor is ligated or incorporated onto the
opposite side of the same RNA molecule of interest in a second reaction,
often with an intervening gel purification, phosphorylation, or reverse
transcription step (see, e.g., Elbashir et al., Genes and Development
15:188-200, 2001; Ambros and Lee, Methods in Mol. Biol. 265:131-58, 2004;
Berezikov et al., Nature Genet. Supp. 38:S2-S7, 2006; Takada et al.,
Nucl. Acids Res. 34(17):e115, 2006; Michael, Methods in Mol. Biol.
342:189-207, 2006; and Takada and Mano, Nature Protocols 2(12):3136-45,
2007). It is to be understood that, with respect to the current
teachings, the order of adding components to the ligation reaction
composition is generally not significant and is intended to be
encompassed within the term "forming a ligation reaction composition" or
similar terms used herein, unless expressly stated otherwise. Thus, the
sequential addition of one adaptor (for example, a first adaptor) to a
reaction composition comprising RNA molecules and a ligase, followed by
the subsequent addition of the other adaptor (in this example, the second
adaptor) to that reaction composition is within the intended scope of the
instant teachings, regardless of whether there is an incubation step
between the addition of one adaptor and the addition of the other
adaptor.

[0135] Reverse Transcription:

[0136] In some embodiments, detecting the RNA molecule comprises reverse
transcribing the ligated product to form a reverse transcribed product.
The terms "reverse transcribing" and "reverse transcription" and forms
thereof as used herein refer to the process of generating a
double-stranded RNA-DNA hybrid molecule, starting with the ligated
product, based on the sequential catalytic addition of
deoxyribonucleotides or analogs of deoxyribonucleotides to the hybrid
molecule in a template dependent manner using a polypeptide having
RNA-directed DNA polymerase transcription activity. According to the
current teachings, the fourth oligonucleotide of the second adaptor can
serve as the primer for reverse transcribing the ligation product to
generate the reverse transcribed product. Addition of a separate primer
for reverse transcription, therefore, is not necessary.

[0138] In some embodiments, the RNA-directed DNA polymerase transcription
activity can be carried out by a DNA-directed DNA polymerase that
possesses RNA-directed DNA polymerase activity under certain reaction
conditions, for example but not limited to, Tth DNA polymerase and DNA
polymerase I from Carboxydothermus hydrogenoformans.

[0139] Ribonuclease H Digestion:

[0140] In some embodiments, the reverse transcribed product is digested
with ribonuclease H to remove at least some of the ribonucleosides to
form an amplification template. The term "digesting", particularly in
reference to a ribonuclease, refers to the catalysis of RNA into smaller
components, for example but not limited to, cleavage of the RNA strand of
the reverse transcribed product by RNase H to generate a single-stranded
or substantially single-stranded cDNA molecule that can serve as an
amplification template of the current teachings.

[0143] Amplification can comprise thermocycling (sometimes referred to as
cycling or thermal cycling) or can be performed isothermally. In certain
embodiments, amplifying comprises at least one cycle, and typically
multiple cycles, of the sequential steps of: hybridizing a primer with a
complementary or substantially complementary sequence of an amplification
template, an amplified product, or the complement of either; synthesizing
a strand of nucleotides in a template-dependent manner using a
polymerase; and denaturing the newly-formed nucleic acid duplex to
separate the strands. The cycle may or may not be repeated, as desired.
In some embodiments, amplifying comprises a cycling the amplification
reaction composition in a thermocycler, for example but not limited to a
GeneAmp® PCR System 9700, 9600, 2700, or 2400 thermocycler (all from
Applied Biosystems). In certain embodiments, newly-formed nucleic acid
duplexes are not initially denatured, but are used in their
double-stranded form in one or more subsequent steps and either or both
strands can, but need not, serve as a surrogate for the corresponding RNA
molecule of interest. In certain embodiments, single-stranded amplicons
are generated, for example but not limited to asymmetric PCR,
asynchronous PCR, or transcription.

[0144] Primer extension is an amplifying technique that comprises
elongating a primer that is annealed to a template in the 5'=>3'
direction using an extending enzyme such as a polymerase to form an
extension product, for example but not limited to reverse transcribing a
ligated product or amplifying an amplification template or an amplified
product. According to certain embodiments, with appropriate buffers,
salts, pH, temperature, and nucleotide triphosphates, a polymerase
incorporates nucleotides complementary to the template strand starting at
the 3'-end of an annealed primer, to generate a complementary strand. In
certain embodiments, the polymerase used for primer extension lacks or
substantially lacks 5'-exonuclease activity.

[0145] In some embodiments, the amplification template is combined with at
least one forward primer, at least one reverse primer, and a polypeptide
having DNA-directed DNA polymerase activity to form an amplification
reaction composition.

[0146] The term "DNA polymerase" is used in a broad sense herein and
refers to any polypeptide that is able to catalyze the addition of
deoxyribonucleotides or analogs of deoxyribonucleotides to a nucleic acid
polymer in a template dependent manner for example, but not limited to,
the sequential addition of deoxyribonucleotides to the 3'-end of a primer
that is annealed to a nucleic acid template during a primer extension
reaction. Typically DNA polymerases include DNA-directed DNA polymerases
and RNA-directed DNA polymerases, including reverse transcriptases. Some
reverse transcriptases possess DNA-directed DNA polymerase activity under
certain reaction conditions, including AMV reverse transcriptase and MMLV
reverse transcriptase. Some DNA-directed DNA polymerases possess reverse
transcriptase under certain reaction conditions, for example, but not
limited to Thermus thermophilus (Tth) DNA polymerase. Descriptions of DNA
polymerases can be found in, among other places, Lehninger Principles of
Biochemistry, 3d ed., Nelson and Cox, Worth Publishing, New York, N.Y.,
2000, particularly Chapters 26 and 29; Twyman, Advanced Molecular
Biology: A Concise Reference, Bios Scientific Publishers, New York, N.Y.,
1999; Ausubel et al., Current Protocols in Molecular Biology, John Wiley
& Sons, Inc., including supplements through May 2005 (hereinafter
"Ausubel et al."); Lin and Jaysena, J. Mol. Biol. 271:100-11, 1997;
Pavlov et al., Trends in Biotechnol. 22:253-60, 2004; and Enzymatic
Resource Guide: Polymerases, 1998, Promega, Madison, Wis. Expressly
within the intended scope of the terms DNA-directed DNA polymerase and
RNA-directed DNA polymerase are enzymatically active mutants or variants
thereof, including enzymes modified to confer different
temperature-sensitive properties (see, e.g., U.S. Pat. Nos. 5,773,258;
5,677,152; and 6,183,998; and DNA Amplification: Current Techniques and
Applications, Demidov and Broude, eds., Horizon Bioscience, 2004,
particularly in Chapter 1.1).

[0147] Enzymatically Active Mutants or Variants of Enzymes:

[0148] For the purposes of the current teachings, when a specific enzyme
or a polypeptide comprising enzymatic activity is described or claimed,
enzymatically active mutants or variants of that enzyme/polypeptide are
intended to be included, unless specifically stated otherwise. For
illustration purposes but not as a limitation, when the terms "Rnl2" or
"Rnl2 ligase" are used in this specification or the appended claims, the
naturally-occurring or wild-type Rnl2 ligase as well as all enzymatically
active mutants or variants of Rnl2 ligase are intended to be included,
unless specifically stated otherwise. Similarly, an RNA-directed DNA
polymerase, a ribonuclease, or a DNA-directed DNA polymerase, is
considered an equivalent to an enzymatically active mutant or variant
thereof. The term "enzymatically active mutant or variant thereof,"
refers to one or more polypeptides derived from the corresponding enzyme
that retains at least some of the desired enzymatic activity, such as
ligating, reverse transcribing, digesting, amplifying, or as appropriate.
Also within the scope of this term are: enzymatically active fragments,
including but not limited to, cleavage products, for example but not
limited to Klenow fragment, Stoffel fragment, or recombinantly expressed
fragments and/or polypeptides that are smaller in size than the
corresponding enzyme; mutant forms of the corresponding enzyme, including
but not limited to, naturally-occurring mutants, such as those that vary
from the "wild-type" or consensus amino acid sequence, mutants that are
generated using physical and/or chemical mutagens, and genetically
engineered mutants, for example but not limited to random and
site-directed mutagenesis techniques; amino acid insertions and
deletions, truncated forms, and changes due to nucleic acid nonsense
mutations, missense mutations, and frameshift mutations (see, e.g.,
Sriskanda and Shuman, Nucl. Acids Res. 26(2):525-31, 1998; Odell et al.,
Nucl. Acids Res. 31(17):5090-5100, 2003); reversibly modified nucleases,
ligases, and polymerases, for example but not limited to those described
in U.S. Pat. No. 5,773,258; biologically active polypeptides obtained
from gene shuffling techniques (see, e.g., U.S. Pat. Nos. 6,319,714 and
6,159,688), splice variants, both naturally occurring and genetically
engineered, provided that they are derived, at least in part, from one or
more corresponding enzymes; polypeptides corresponding at least in part
to one or more such enzymes that comprise modifications to one or more
amino acids of the native sequence, including without limitation, adding,
removing or altering glycosylation, disulfide bonds, hydroxyl side
chains, and phosphate side chains, or crosslinking, provided such
modified polypeptides retain at least some of the desired catalytic
activity; and the like. Expressly within the meaning of the term
"enzymatically active mutants or variants thereof" when used in reference
to a particular enzyme(s) are enzymatically active mutants of that
enzyme, enzymatically active variants of that enzyme, or enzymatically
active mutants of that enzyme and enzymatically active variants of that
enzyme.

[0149] The skilled artisan will readily be able to measure enzymatic
activity using an appropriate assay known in the art. Thus, an
appropriate assay for polymerase catalytic activity might include, for
example, measuring the ability of a variant to incorporate, under
appropriate conditions, rNTPs or dNTPs into a nascent polynucleotide
strand in a template-dependent manner. Likewise, an appropriate assay for
ligase catalytic activity might include, for example, the ability to
ligate adjacently hybridized oligonucleotides comprising appropriate
reactive groups, such as disclosed herein. Protocols for such assays may
be found, among other places, in Sambrook et al., Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press (1989) (hereinafter "Sambrook
et al."), Sambrook and Russell, editors, Molecular Cloning, Vol 3, 3rd
edition, Cold Spring Harbor Press (2001), Ausubel et al., and Housby and
Southern, Nucl. Acids Res. 26:4259-66, 1998) and the references cited
below for the family of Rnl2 ligases.

[0150] Amplification Primers:

[0151] The term "primer" refers to a polynucleotide that selectively
hybridizes to a corresponding primer-binding site of an amplification
template, an amplified product, or both; and allows the synthesis of a
sequence complementary to the corresponding polynucleotide template from
its 3' end. A "primer pair" comprises a forward primer and a reverse
primer that anneal to one strand of an amplification product or its
complement. Primer pairs are particularly useful in certain exponential
amplification techniques, such as the polymerase chain reaction. In
certain embodiments, a forward primer and the corresponding reverse
primer of a primer pair have different melting temperatures (Tm) to
permit asynchronous primer PCR.

[0152] As used herein, "forward" and "reverse" are used to indicate
relative orientation of primers on a polynucleotide sequence such as an
amplification template or an amplified product. For illustration purposes
but not as a limitation, consider a single-stranded polynucleotide drawn
in a horizontal, left to right orientation with its 5'-end on the left.
The "reverse" primer is designed to anneal with the downstream
primer-binding site at or near the "3'-end" of this illustrative
polynucleotide in a 5' to 3' orientation, right to left. The
corresponding "forward" primer is designed to anneal with the complement
of the upstream primer-binding site at or near the "5'-end" of the
polynucleotide in a 5' to 3' "forward" orientation, left to right. Thus,
the reverse primer comprises a sequence that is complementary to the
"reverse" or downstream primer-binding site of the polynucleotide and the
forward primer comprises a sequence that is the same as the forward or
upstream primer-binding site. It is to be understood that the terms
"3-end" and "5'-end" as used in this paragraph are illustrative only and
do not necessarily refer literally to the respective ends of the
polynucleotide, as such primer-binding sites may be located internally.
Rather, the only limitation is that the reverse primer of this exemplary
primer pair anneals with a reverse primer-binding site that is downstream
or to the right of the forward primer-binding site that comprises the
same sequence as the corresponding forward primer. As will be recognized
by those of skill in the art, these terms are not intended to be
limiting, but rather to provide illustrative orientation in a given
embodiment.

[0153] A primer may comprise a nucleotide sequence of an adaptor
oligonucleotide or a nucleotide sequence corresponding to an adaptor
oligonucleotide. For example, forward primer having SEQ ID NO:5 comprises
the sequence of first oligonucleotide having SEQ ID NO:1 (with T's
instead of U's). Some embodiments of relationships between primers and
adaptor sequences can be understood by the schematic of FIG. 15 in which
arrows depict variously, forward and reverse PCR primers or forward and
reverse SYBR primers. P1 and P2 refer to primer portions.

[0154] As used herein, the term "primer-binding site" refers to a region
of a polynucleotide sequence that can serve directly, or by virtue of its
complement, as the template upon which a primer can anneal for any of a
variety of primer nucleotide extension reactions known in the art (for
example, PCR). It will be appreciated by those of skill in the art that
when two primer-binding sites are present on a single polynucleotide (for
example but not limited to a first extension product or a second
extension product), the orientation of the two primer-binding sites is
generally different. For example, one primer of a primer pair is
complementary to and can hybridize with to the first primer-binding site,
while the corresponding primer of the primer pair is designed to
hybridize with the complement of the second primer-binding site. Stated
another way, in some embodiments the first primer-binding site can be in
a sense orientation, and the second primer-binding site can be in an
antisense orientation. In addition, "universal" primers and
primer-binding sites as used herein are generally chosen to be as unique
as possible given the particular assays and host genomes to ensure
specificity of the assay.

[0155] In some embodiments, a primer and/or an amplified product comprises
a "promoter sequence", including without limitation a sequence suitable
for initiating transcription using a suitable polymerase, for example but
not limited to, T3 RNA polymerase, T7 RNA polymerase, or SP6 RNA
polymerase. Some embodiments of the current teachings employ a
"promoter-primer" in a method of incorporating a promoter sequence into
an amplification product. In some embodiments, a promoter sequence
comprises a multiplicity of different sequences suitable for binding an
RNA polymerase, for example but not limited to a first sequence suitable
for binding a first RNA polymerase and a second sequence suitable for
binding a second RNA polymerase. Those in the art understand that as an
amplification product comprising a promoter sequence is amplified by
certain amplification methods, the complement of the promoter sequence
may be synthesized in the complementary amplicon. Thus, it is to be
understood that the complement of a promoter sequence is expressly
included within the intended meaning of the term promoter sequence, as
used herein. Some embodiments of the disclosed methods and kits employ a
"promoter-primer" in methods of incorporating a desired promoter sequence
into an amplification product.

[0156] Those in the art understand that as an amplified product is
amplified by certain amplification methods, the complement of the
primer-binding site is synthesized in the complementary amplicon. Thus,
it is to be understood that the complement of a primer-binding site is
expressly included within the intended meaning of the term primer-binding
site, as used herein.

[0157] In some embodiments, the amplification methods of the current
teachings comprise a Q-PCR reaction. The terms "quantitative PCR", "real
time PCR", or "Q-PCR" refer to a variety of methods used to quantify the
results of the polymerase chain reaction for specific nucleic acid
sequences. Such methods typically are categorized as kinetics-based
systems that generally determine or compare the amplification factor,
such as determining the threshold cycle (CT), or as co-amplification
methods, that generally compare the amount of product generated from
simultaneous amplification of target and standard templates. Many Q-PCR
techniques comprise reporter probes, intercalating agents, or both. For
example but not limited to TaqMan® probes (Applied Biosystems),
i-probes, molecular beacons, Eclipse probes, scorpion primers, Lux®
primers, FRET primers, ethidium bromide, SYBR® Green I (Molecular
Probes), and PicoGreen® (Molecular Probes). In some embodiments,
detecting comprises a real-time detection instrument. Exemplary real-time
instruments include, the ABI PRISM® 7000 Sequence Detection System,
the ABI PRISM® 7700 Sequence Detection System, the Applied Biosystems
7300 Real-Time PCR System, the Applied Biosystems 7500 Real-Time PCR
System, the Applied Biosystems 7900 HT Fast Real-Time PCR System (all
from Applied Biosystems); the LightCycler® System (Roche Molecular);
the Mx3000P® Real-Time PCR System, the Mx3005P® Real-Time PCR
System, and the Mx4000® Multiplex Quantitative PCR System
(Stratagene, La Jolla, Calif.); and the Smart Cycler System (Cepheid,
distributed by Fisher Scientific). Descriptions of real-time instruments
can be found in, among other places, their respective manufacturer's
users manuals; McPherson; DNA Amplification: Current Technologies and
Applications, Demidov and Broude, eds., Horizon Bioscience, 2004; and
U.S. Pat. No. 6,814,934.

[0158] In certain embodiments, an amplification reaction comprises
multiplex amplification, in which a multiplicity of different
amplification templates, a multiplicity of different amplification
product species, or both, are simultaneously amplified using a
multiplicity of different primer pairs (see, e.g., Henegariu et al.,
BioTechniques 23:504-11, 1997; and Rapley, particularly in Chapter 79).
Certain embodiments of the disclosed methods comprise a single-plex
amplification reaction, including without limitation, an amplification
reaction comprising a multiplicity of single-plex amplifications
performed in parallel, for example but not limited to certain TaqMan®
Array configurations wherein approximately 100 nuclease assays are
performed in parallel to determine whether specific amplified products
are present and in what quantity.

[0159] In certain embodiments, an amplifying reaction comprises asymmetric
PCR. According to certain embodiments, asymmetric PCR comprises an
amplification composition comprising (i) at least one primer pair in
which there is an excess of one primer, relative to the corresponding
primer of the primer pair, for example but not limited to a five-fold, a
ten-fold, or a twenty-fold excess; (ii) at least one primer pair that
comprises only a forward primer or only a reverse primer; (iii) at least
one primer pair that, during given amplification conditions, comprises a
primer that results in amplification of one strand and a corresponding
primer that is disabled; or (iv) at least one primer pair that meets the
description of both (i) and (iii) above. Consequently, when an
amplification template or an amplification product is amplified, an
excess of one strand of the subsequent amplification product (relative to
its complement) is generated. Descriptions of asymmetric PCR, can be
found in, among other places, McPherson, particularly in Chapter 5; and
Rapley, particularly in Chapter 64.

[0160] In certain embodiments, one may use at least one primer pair
wherein the melting temperature (Tm50) of one of the primers is
higher than the Tm50 of the other primer, sometimes referred to as
asynchronous primer PCR (A-PCR, see, e.g., U.S. Pat. No. 6,887,664). In
certain embodiments, the Tm50 of the forward primer is at least
4-15° C. different from the Tm50 of the corresponding reverse
primer. In certain embodiments, the Tm50 of the forward primer is at
least 8-15° C. different from the Tm50 of the corresponding
reverse primer. In certain embodiments, the Tm50 of the forward
primer is at least 10-15° C. different from the Tm50 of the
corresponding reverse primer. In certain embodiments, the Tm50 of
the forward primer is at least 10-12° C. different from the
Tm50 of the corresponding reverse primer. In certain embodiments, in
at least one primer pair, the Tm50 of a forward primer differs from
the Tm50 of the corresponding reverse primer by at least about
4° C., by at least about 8° C., by at least about
10° C., or by at least about 12° C.

[0161] In certain amplifying embodiments, in addition to the difference in
Tm50 of the primers in a primer pair, there is also an excess of one
primer relative to the other primer in the primer pair. In certain
embodiments, there is a five- to twenty-fold excess of one primer
relative to the other primer in the primer pair. In certain embodiments
of A-PCR, the primer concentration is at least 50 nM.

[0162] In A-PCR according to certain embodiments, one may use conventional
PCR in the first cycles of amplification such that both primers anneal
and both strands of a double-stranded amplicon are amplified. By raising
the temperature in subsequent cycles of the same amplification reaction,
however, one may disable the primer with the lower Tm such that only
one strand is amplified. Thus, the subsequent cycles of A-PCR in which
the primer with the lower Tm is disabled result in asymmetric
amplification. Consequently, when the target region or an amplification
product is amplified, an excess of one strand of the subsequent
amplification product (relative to its complement) is generated.

[0163] According to certain embodiments of A-PCR, the level of
amplification can be controlled by changing the number of cycles during
the first phase of conventional PCR cycling. In such embodiments, by
changing the number of initial conventional cycles, one may vary the
amount of the double-stranded amplification products that are subjected
to the subsequent cycles of PCR at the higher temperature in which the
primer with the lower Tm is disabled.

[0164] In certain embodiments, amplifying comprises in vitro
transcription. In some embodiments, a first adaptor, a second adaptor, a
first primer, a second primer, or combinations thereof, comprise a
promoter sequence or its complement, for example but not limited to, a
promoter-primer. In some embodiments, a reverse transcribed product
comprising a promoter or an amplified product comprising a promoter is
combined with ribonucleotide triphosphates, an appropriate buffer system,
and a suitable RNA polymerase, for example but not limited to, SP6, T3,
or T7 RNA polymerase and amplified RNA (aRNA) are generated according to
known methods. The aRNA may be used for array analysis, such as
microarray or bead array analysis, wherein the sequence and quantity of
the aRNA species can be determined. Thus, in certain embodiments, such
aRNA serves as a surrogate for the corresponding RNA molecule.

[0165] Certain methods of optimizing amplification reactions are known to
those skilled in the art. For example, it is known that PCR may be
optimized by altering times and temperatures for annealing,
polymerization, and denaturing, as well as changing the buffers, salts,
and other reagents in the reaction composition. Optimization may also be
affected by the design of the primers used. For example, the length of
the primers, as well as the G-C:A-T ratio may alter the efficiency of
primer annealing, thus altering the amplification reaction. Descriptions
of amplification optimization can be found in, among other places, James
G. Wetmur, "Nucleic Acid Hybrids, Formation and Structure," in Molecular
Biology and Biotechnology, pp. 605-8, (Robert A. Meyers ed., 1995);
McPherson, particularly in Chapter 4; Rapley; and Protocols &
Applications Guide, rev. 9/04, Promega Corp., Madison, Wis.

[0166] Purifying the amplified product according to the present teachings
comprises any process that removes at least some unligated adaptors,
unligated RNA molecules, byproducts, primers, enzymes or other components
of the ligation reaction composition, the amplification reaction
composition, or both following at least one cycle of amplification. Such
processes include, but are not limited to, molecular weight/size
exclusion processes, e.g., gel filtration chromatography or dialysis,
sequence-specific hybridization-based pullout methods, affinity capture
techniques, precipitation, adsorption, gel electrophoresis, conventional
cloning, conventional cloning with concatamerization, or other nucleic
acid purification techniques. In some embodiments purifying the amplified
product comprises gel electrophoresis, including without limitation,
polyacrylamide gel electrophoresis (PAGE) and/or agarose gel
electrophoresis. In certain embodiments, the amplified product is
purified using high-performance liquid chromatography (HPLC; sometimes
also referred to as high-pressure liquid chromatography).

[0167] Detection:

[0168] The RNA molecule of interest is detected by detecting the ligated
product or a surrogate thereof. In some embodiments, the ligated product
is reverse transcribed as described above and the reverse transcribed
product is placed on an array and detected using standard methods known
by one of skill in the art. In some embodiments, the reverse transcribed
product is labeled with biotin and detection is by using streptavidin
binding thereto. In some embodiments, the reverse transcribed product is
purified using glass fiber filters, beads or is gel-purified. In some
embodiments, the reverse transcribed product is combined with a peptide
comprising ribonuclease activity to form a digestion reaction composition
and incubated under conditions suitable for digesting at least some of
the ribonucleosides from the reverse transcribed product to form an
amplification template.

[0169] The terms "detecting" and "detection" are used in a broad sense
herein and encompass any technique by which one can determine whether or
not a particular RNA molecule i.e., an RNA molecule of interest, is
present in a sample. In some embodiments, the presence of a surrogate is
detected, directly or indirectly, allowing the presence or absence of the
corresponding RNA molecule to be determined. For example, the presence of
a surrogate is detected by detecting a family of labeled sequencing
products obtained using an amplified product or a ligated product as the
template; or detecting the fluorescence generated when a nuclease
reporter probe, annealed to an amplified product, is cleaved by a
polymerase, wherein the detectable signal or detectable change in signal
indicates that the corresponding amplified product and/or ligated product
has been amplified and thus the corresponding RNA molecule is present in
the sample. In some embodiments, detecting comprises quantitating the
detectable signal, including without limitation, a real-time detection
method, such as quantitative PCR ("O-PCR"). In some embodiments,
detecting comprises determining the sequence of a sequencing product or a
family of sequencing products generated using an amplification product as
the template; in some embodiments, such detecting comprises obtaining the
sequence of a family of sequencing products. In some embodiments,
detecting an RNA molecule comprises a nucleic acid dye, for example but
not limited to, in a Q-PCR reaction composition. Those in the art will
understand that the ligated products, reverse transcribed products,
amplification templates, and amplified sequences each serve as a
surrogate for the RNA molecule from which they were directly or
indirectly generated and that by detecting any of these products one is
directly or indirectly detecting the corresponding RNA molecule.

[0170] The term "reporter probe" refers to a sequence of nucleotides,
nucleotide analogs, or nucleotides and nucleotide analogs, that
specifically anneals with a corresponding amplicon, for example but not
limited to a PCR product, and when detected, including but not limited to
a change in intensity or of emitted wavelength, is used to identify,
detect, and/or quantify the corresponding amplicon and thus the
corresponding RNA molecule. Thus, by indirectly detecting the amplicon,
one can determine that the corresponding RNA molecule is present in the
sample. Most reporter probes can be categorized based on their mode of
action, for example but not limited to: nuclease probes, including
without limitation TaqMan® probes; extension probes including without
limitation scorpion primers, Lux® primers, Amplifluors, and the like;
and hybridization probes including without limitation molecular beacons,
Eclipse® probes, light-up probes, pairs of singly-labeled reporter
probes, hybridization probe pairs, and the like. In certain embodiments,
reporter probes comprise an amide bond, a locked nucleic acid (LNA), a
universal base, or combinations thereof, and can include stem-loop and
stem-less reporter probe configurations. Certain reporter probes are
singly-labeled, while other reporter probes are doubly-labeled. Dual
probe systems that comprise FRET between adjacently hybridized probes are
within the intended scope of the term reporter probe. In certain
embodiments, a reporter probe comprises a fluorescent reporter group and
a quencher (including without limitation dark quenchers and fluorescent
quenchers). Some non-limiting examples of reporter probes include
TaqMan® probes; Scorpion probes (also referred to as scorpion
primers); Lux® primers; FRET primers; Eclipse® probes; molecular
beacons, including but not limited to FRET-based molecular beacons,
multicolor molecular beacons, aptamer beacons, PNA beacons, and antibody
beacons; labeled PNA clamps, labeled PNA openers, labeled LNA probes, and
probes comprising nanocrystals, metallic nanoparticles and similar hybrid
probes (see, e.g., Dubertret et al., Nature Biotech. 19:365-70, 2001;
Zelphati et al., BioTechniques 28:304-15, 2000). In certain embodiments,
reporter probes further comprise minor groove binders including but not
limited to TaqMan®MGB probes and TaqMan®MGB-NFQ probes (both from
Applied Biosystems). In certain embodiments, reporter probe detection
comprises fluorescence polarization detection (see, e.g., Simeonov and
Nikiforov, Nucl. Acids Res. 30:e91, 2002).

[0171] The term "reporter group" is used in a broad sense herein and
refers to any identifiable tag, label, or moiety. The ordinarily skilled
artisan will appreciate that many different species of reporter groups
can be used in the present teachings, either individually or in
combination with one or more different reporter group. In certain
embodiments, a reporter group emits a fluorescent, a chemiluminescent, a
bioluminescent, a phosphorescent, or an electrochemiluminescent signal.
Some non-limiting examples of reporter groups include fluorophores,
radioisotopes, chromogens, enzymes, antigens including but not limited to
epitope tags, semiconductor nanocrystals such as quantum dots, heavy
metals, dyes, phosphorescence groups, chemiluminescent groups,
electrochemical detection moieties, binding proteins, phosphors, rare
earth chelates, transition metal chelates, near-infrared dyes,
electrochemiluminescence labels, and mass spectrometer-compatible
reporter groups, such as mass tags, charge tags, and isotopes (see, e.g.,
Haff and Smirnov, Nucl. Acids Res. 25:3749-50, 1997; Xu et al., Anal.
Chem. 69:3595-3602, 1997; Sauer et al., Nucl. Acids Res. 31:e63, 2003).

[0172] The term reporter group also encompasses an element of
multi-element reporter systems, including without limitation, affinity
tags such as biotin:avidin, antibody:antigen, and the like, in which one
element interacts with one or more other elements of the system in order
to effect the potential for a detectable signal. Some non-limiting
examples of multi-element reporter systems include an oligonucleotide
comprising a biotin reporter group and a streptavidin-conjugated
fluorophore, or vice versa; an oligonucleotide comprising a DNP reporter
group and a fluorophore-labeled anti-DNP antibody; and the like. Detailed
protocols for attaching reporter groups to nucleic acids can be found in,
among other places, Hermanson, Bioconjugate Techniques, Academic Press,
San Diego, 1996; Current Protocols in Nucleic Acid Chemistry, Beaucage et
al., eds., John Wiley & Sons, New York, N.Y. (2000), including
supplements through April 2005; and Haugland, Handbook of Fluorescent
Probes and Research Products, 9th ed., Molecular Probes, 2002.

[0173] Multi-element interacting reporter groups are also within the
intended scope of the term reporter group, such as fluorophore-quencher
pairs, including without limitation fluorescent quenchers and dark
quenchers (also known as non-fluorescent quenchers). A fluorescent
quencher can absorb the fluorescent signal emitted from a fluorophore and
after absorbing enough fluorescent energy, the fluorescent quencher can
emit fluorescence at a characteristic wavelength, e.g., fluorescent
resonance energy transfer (FRET). For example without limitation, the
FAM®-TAMRA® dye pair can be illuminated at 492 nm, the excitation
peak for FAM® dye, and emit fluorescence at 580 nm, the emission peak
for TAMRA® dye. A dark quencher, appropriately paired with a
fluorescent reporter group, absorbs the fluorescent energy from the
fluorophore, but does not itself fluoresce. Rather, the dark quencher
dissipates the absorbed energy, typically as heat. Some non-limiting
examples of dark or nonfluorescent quenchers include Dabcyl, Black Hole
Quenchers, Iowa Black, QSY-7, AbsoluteQuencher, Eclipse®
non-fluorescent quencher, metal clusters such as gold nanoparticles, and
the like. Certain dual-labeled probes comprising fluorophore-quencher
pairs can emit fluorescence when the members of the pair are physically
separated, for example but without limitation, nuclease probes such as
TaqMan® probes. Other dual-labeled probes comprising
fluorophore-quencher pairs can emit fluorescence when the members of the
pair are spatially separated, for example but not limited to
hybridization probes such as molecular beacons or extension probes such
as Scorpion® primers. Fluorophore-quencher pairs are well known in the
art and used extensively for a variety of reporter probes (see, e.g.,
Yeung et al., BioTechniques 36:266-75, 2004; Dubertret et al., Nat.
Biotech. 19:365-70, 2001; and Tyagi et al., Nat. Biotech. 18:1191-96,
2000).

[0174] The term "nucleic acid dye" as used herein refers to a fluorescent
molecule that is specific for a double-stranded polynucleotide or that
emits a substantially greater fluorescent signal when associated with a
double-stranded polynucleotide than with a single-stranded
polynucleotide. Typically nucleic acid dye molecules associate with
double-stranded segments of polynucleotides by intercalating between the
base pairs of the double-stranded segment, by binding in the major or
minor grooves of the double-stranded segment, or both. Non-limiting
examples of nucleic acid dyes include ethidium bromide, DAPI, Hoechst
derivatives including without limitation Hoechst 33258 and Hoechst 33342,
intercalators comprising a lanthanide chelate (for example but not
limited to a nalthalene diimide derivative carrying two fluorescent
tetradentate β-diketone-Eu3+ chelates (NDI-(BHHCT-Eu3+)2),
see, e.g., Nojima et al., Nucl. Acids Res. Supplement No. 1, 105-06
(2001)), ethidium bromide, and certain unsymmetrical cyanine dyes such as
SYBR® Green, SYBR® Gold, PicoGreen®, and BOXTO.

[0175] In certain embodiments, detecting comprises an instrument, i.e.,
using an automated or semi-automated detecting device that can, but need
not comprise a computer algorithm. In certain embodiments, a detecting
instrument comprises or is coupled to a device for graphically displaying
the intensity of an observed or measured parameter of an extension
product or its surrogate on a graph, monitor, electronic screen, magnetic
media, scanner print-out, or other two- or three-dimensional display
and/or recording the observed or measured parameter. In certain
embodiments, the detecting step is combined with or is a continuation of
at least one separating step, for example but not limited to a capillary
electrophoresis instrument comprising at least one fluorescent scanner
and at least one graphing, recording, or readout component; a
chromatography column coupled with an absorbance monitor or fluorescence
scanner and a graph recorder; a chromatography column coupled with a mass
spectrometer comprising a recording and/or a detection component; or a
microarray with a data recording device such as a scanner or CCD camera.
In certain embodiments, the detecting step is combined with an amplifying
step, for example but not limited to, real-time analysis such as Q-PCR.
Exemplary systems for performing a detecting step include the ABI
PRISM® Genetic Analyzer instrument series, the ABI PRISM® DNA
Analyzer instrument series, the ABI PRISM® Sequence Detection Systems
instrument series, and the Applied Biosystems Real-Time PCR instrument
series (all from Applied Biosystems); and microarrays and related
software such as the Applied Biosystems microarray and Applied Biosystems
1700 Chemiluminescent Microarray Analyzer and other commercially
available microarray and analysis systems available from Affymetrix,
Agilent, among others (see also Gerry et al., J. Mol. Biol. 292:251-62,
1999; De Bellis et al., Minerva Biotec 14:247-52, 2002; and Stears et
al., Nat. Med. 9:140-45, including supplements, 2003) or bead array
platforms (Illumina, San Diego, Calif.). Exemplary software includes
GeneMapper® Software, GeneScan® Analysis Software, and
Genotyper® software (all from Applied Biosystems).

[0176] In certain embodiments, an RNA molecule can be detected and
quantified based on the mass-to-charge ratio (m/z) of at least a part of
an amplified product and/or a ligated product. For example, in some
embodiments, a primer or an adapter comprises a mass
spectrometry-compatible reporter group, including without limitation,
mass tags, charge tags, cleavable portions, or isotopes that are
incorporated into an amplified product and can be used for mass
spectrometer detection (see, e.g., Haff and Smirnov, Nucl. Acids Res.
25:3749-50, 1997; and Sauer et al., Nucl. Acids Res. 31:e63, 2003). An
amplified product can be detected by mass spectrometry allowing the
presence or absence of the corresponding RNA molecule to be determined.
In some embodiments, a primer or an adaptor comprises a restriction
enzyme site, a cleavable portion, or the like, to facilitate release of a
part of an amplified product for detection. In certain embodiments, a
multiplicity of amplified products are separated by liquid chromatography
or capillary electrophoresis, subjected to ESI or to MALDI, and detected
by mass spectrometry. Descriptions of mass spectrometry can be found in,
among other places, The Expanding Role of Mass Spectrometry in
Biotechnology, Gary Siuzdak, MCC Press, 2003.

[0177] In certain embodiments, surrogates such as a reporter probe or a
cleaved portion of a reporter probe are detected, directly or indirectly.
For example but not limited to, hybridizing an amplified product to a
reporter probe comprising a quencher, including without limitation, a
molecular beacon, including stem-loop and stem-free beacons, a
TaqMan® probe or other nuclease probe, a LightSpeed® PNA probe, or
a microarray capture probe. In certain embodiments, the hybridization
occurs when the molecular beacon and the amplified product are free in
solution and a detectable signal or a detectably different signal is
emitted. In other embodiments, an amplified product hybridizes to or is
bound to a solid surface such as a microarray and a detectable signal or
a detectably different signal is emitted (see, e.g., EviArrays® and
EviProbes®, Evident Technologies).

[0178] In certain embodiments, detecting comprises measuring or
quantifying the detectable signal of a reporter group or the change in a
detectable signal of a reporter group, typically due to the presence of
an amplified product. For illustration purposes but not as a limitation,
an unhybridized reporter probe may emit a low level, but detectable
signal that quantitatively increases when hybridized with the amplified
product, including without limitation, certain molecular beacons, LNA
probes, PNA probes, and light-up probes (see, e.g., Svanik et al.,
Analyt. Biochem. 281:26-35, 2000; Nikiforov and Jeong, Analyt. Biochem.
275:248-53, 1999; and Simeonov and Nikiforov, Nucl. Acids Res. 30:e91,
2002). In certain embodiments, detecting comprises measuring fluorescence
polarization.

[0179] In some embodiments, determining whether a particular RNA molecule
is present in a sample comprises evaluating an internal standard or a
control sequence, such as a standard curve for the corresponding target
region, an internal size standard, or combinations thereof. In some
embodiments, a control sequence or an internal reference dye is employed
to account for lane-to-lane, capillary-to-capillary, and/or
assay-to-assay variability. In certain embodiments, an internal control
sequence comprises an unrelated nucleic acid that is amplified in
parallel to validate the amplification reaction or the detection
technique.

[0180] Those in the art understand that the detection techniques employed
are generally not limiting. Rather, a wide variety of detection methods
are within the scope of the disclosed methods and kits, provided that
they allow the presence or absence of an RNA molecule in the sample to be
determined.

[0181] In some embodiments, the disclosed methods and kits comprise a
microfluidics device, "lab on a chip", or micrototal analytical system
(μTAS). In some embodiments, sample preparation is performed using a
microfluidics device. In some embodiments, an amplification reaction is
performed using a microfluidics device. In some embodiments, a sequencing
or Q-PCR reaction is performed using a microfluidic device. In some
embodiments, the nucleotide sequence of at least a part of an amplified
product is obtained using a microfluidics device. In some embodiments,
detecting comprises a microfluidic device, including without limitation,
a TaqMan® Low Density Array (Applied Biosystems). Descriptions of
exemplary microfluidic devices can be found in, among other places,
Published PCT Application Nos. WO/0185341 and WO 04/011666; Kartalov and
Quake, Nucl. Acids Res. 32:2873-79, 2004; and Fiorini and Chiu,
BioTechniques 38:429-46, 2005.

[0182] Sequencing:

[0183] In some embodiments, the sequence of at least part of the amplified
product is determined thereby detecting the RNA molecule of interest. The
term "sequencing" is used in a broad sense herein and refers to any
technique known in the art that allows the order of at least some
consecutive nucleotides in at least part of a RNA to be identified,
including without limitation at least part of an extension product or a
vector insert. Some non-limiting examples of sequencing techniques
include Sanger's dideoxy terminator method and the chemical cleavage
method of Maxam and Gilbert, including variations of those methods;
sequencing by hybridization, for example but not limited to,
hybridization of amplified products to a microarray or a bead, such as a
bead array; pyrosequencing (see, e.g., Ronaghi et al., Science
281:363-65, 1998); and restriction mapping. Some sequencing methods
comprise electrophoreses, including without limitation capillary
electrophoresis and gel electrophoresis; mass spectrometry; and single
molecule detection. In some embodiments, sequencing comprises direct
sequencing, duplex sequencing, cycle sequencing, single-base extension
sequencing (SBE), solid-phase sequencing, or combinations thereof. In
some embodiments, sequencing comprises an detecting the sequencing
product using an instrument, for example but not limited to an ABI
PRISM® 377 DNA Sequencer, an ABI PRISM® 310, 3100, 3100-Avant,
3730, or 3730×1 Genetic Analyzer, an ABI PRISM® 3700 DNA
Analyzer, or an Applied Biosystems SOLiD® System (all from Applied
Biosystems), a Genome Sequencer 20 System (Roche Applied Science), or a
mass spectrometer. In certain embodiments, sequencing comprises emulsion
PCR (see, e.g., Williams et al., Nature Methods 3(7):545-50, 2006.) In
certain embodiments, sequencing comprises a high throughput sequencing
technique, for example but not limited to, massively parallel signature
sequencing (MPSS). Descriptions of MPSS can be found, among other places,
in Zhou et al., Methods of Molecular Biology 331:285-311, Humana Press
Inc.; Reinartz et al., Briefings in Functional Genomics and Proteomics,
1:95-104, 2002; Jongeneel et al., Genome Research 15:1007-14, 2005. In
some embodiments, sequencing comprises incorporating a dNTP, including
without limitation a dATP, a dCTP, a dGTP, a dTTP, a dUTP, a dITP, or
combinations thereof and including dideoxyribonucleotide versions of
dNTPs, into an amplified product.

[0184] Further exemplary techniques that are useful for determining the
sequence of at least a portion of a nucleic acid molecule include,
without limitation, emulsion-based PCR followed by any suitable massively
parallel sequencing or other high-throughput technique. In some
embodiments, determining the sequence of at least a part of an amplified
product to detect the corresponding RNA molecule comprises quantitating
the amplified product. In some embodiments, sequencing is carried out
using the SOLiD® System (Applied Biosystems) as described in, for
example, PCT patent application publications WO 06/084132 entitled
"Reagents, Methods, and Libraries For Bead-Based Sequencing and
WO07/121,489 entitled "Reagents, Methods, and Libraries for Gel-Free
Bead-Based Sequencing." In some embodiments, quantitating the amplified
product comprises real-time or end-point quantitative PCR or both. In
some embodiments, quantitating the amplified product comprises generating
an expression profile of the RNA molecule to be detected, such as an mRNA
expression profile or a miRNA expression profile. In certain embodiments,
quantitating the amplified product comprises one or more 5'-nuclease
assays, for example but not limited to, TaqMan® Gene Expression
Assays and TaqMan® miRNA Assays, which may comprise a microfluidics
device including without limitation, a low density array. Any suitable
expression profiling technique known in the art may be employed in
various embodiments of the disclosed methods.

[0185] Those in the art will appreciate that the sequencing method
employed is not typically a limitation of the present methods. Rather,
any sequencing technique that provides the order of at least some
consecutive nucleotides of at least part of the corresponding amplified
product or RNA to be detected or at least part of a vector insert derived
from an amplified product can typically be used in the current methods.
Descriptions of sequencing techniques can be found in, among other
places, McPherson, particularly in Chapter 5; Sambrook and Russell;
Ausubel et al.; Siuzdak, The Expanding Role of Mass Spectrometry in
Biotechnology, MCC Press, 2003, particularly in Chapter 7; and Rapley. In
some embodiments, unincorporated primers and/or dNTPs are removed prior
to a sequencing step by enzymatic degradation, including without
limitation exonuclease I and shrimp alkaline phosphatase digestion, for
example but not limited to the ExoSAP-IT® reagent (USB Corporation).
In some embodiments, unincorporated primers, dNTPs, and/or ddNTPs are
removed by gel or column purification, sedimentation, filtration, beads,
magnetic separation, or hybridization-based pull out, as appropriate
(see, e.g., ABI PRISM® Duplex® 384 Well F/R Sequence Capture Kit,
Applied Biosystems P/N 4308082).

[0186] Those in the art will appreciate that, in certain embodiments, the
read length of the sequencing/resequencing technique employed may be a
factor in the size of the RNA molecules that can effectively be detected
(see, e.g., Kling, Nat. Biotech. 21(12):1425-27). In some embodiments,
the amplified products generated from the RNA molecules from a first
sample are labeled with a first identification sequence (sometimes
referred to as a "barcode" herein) or other marker, the amplified
products generated from the RNA molecules from a second sample are
labeled with a second identification sequence or second marker, and the
amplified products comprising the first identification sequence and the
amplified products comprising the second identification sequence are
pooled prior to determining the sequence of the corresponding RNA
molecules in the corresponding samples. In certain embodiments, three or
more different RNA libraries, each comprising a identifier sequence that
is specific to that library, are combined. In some embodiments, a first
adaptor, a second adaptor, a forward primer, a reverse primer, or
combinations thereof, comprise an identification sequence or the
complement of an identification sequence. In certain embodiments, the
identification sequence comprises one of (i) 5'-AAGCCC, (ii) 5'-CACACC,
(iii) 5'-CCCCTT, (iv) 5'-CATCGG, (v) 5-TCGTTG, (vi) 5'-GGGCAC, (vii)
5'-CCAGAC, (viii) 5'-CTCCGT, (ix) 5'-CCCTTC, (x) 5'-GCGGTC, or the
complement of any one of these sequences (i)-(x). In some embodiments, a
reverse primer comprises a sequence of SEQ ID NO:6 or SEQ ID NO:15 to SEQ
ID NO:23 as described in Example 11.

[0187] Libraries:

[0188] The present teachings provide compositions, methods and kits for
detecting an RNA molecule. According to certain embodiments, a library
comprising a multiplicity of different amplified product species is
generated wherein at least one species of amplified product corresponds
to one species of small RNA present in the sample.

[0189] According to certain illustrative embodiments of the instant
teachings, for example, a sample comprising a multiplicity of small RNA
species, a multiplicity of mRNA species, or both, is combined with a
multiplicity of different first adaptor species, a multiplicity of
different second adaptor species, and a polypeptide comprising
double-strand specific RNA ligase activity to form a ligation reaction
composition. In some embodiments, the mRNA is fragmented, depleted of
undesired nucleic acid species (for example but not limited to, rRNA,
high copy number mRNAs or genomic DNA), or depleted and fragmented. The
ligation reaction composition is incubated under conditions suitable for
at least some of the adaptor species to hybridize with corresponding RNA
molecules. It is to be understood that the process of (i) combining the
adaptor species with the sample containing RNA, (ii) incubating to allow
the adaptors to anneal with a corresponding RNA molecule, then (iii)
adding the ligase to the reaction composition is within the intended
scope of forming the ligation reaction composition and incubating, unless
expressly stated otherwise.

[0190] The multiplicity of different adaptor species typically comprise
sets of RNA/DNA oligonucleotides with single-stranded degenerate sequence
at one end and in certain embodiments, a defined sequence at or near the
other end that may serve as a binding site for amplification primers or
reporter probes, sample identification (for example but not limited to
pooling libraries generated from different starting materials and
subsequently identifying the source of the amplified library), and/or
sequencing of subsequently generated amplified products. In certain
embodiments, hybridizing sample with Adaptor Mix A will yield amplified
products suitable for SOLiD® sequencing from the 5' ends of the
sequence corresponding to the RNA molecule within the amplified product.
Conversely, hybridization with Adaptor Mix B yields amplified products
suitable for SOLiD® sequencing from the 3' ends. A polypeptide
comprising double-strand specific RNA ligase activity is then added to
the mixture to ligate the hybridized adaptors to the small RNA molecules.

[0191] The ligation reaction composition is combined with a DNA polymerase
comprising RNA-dependent DNA polymerase activity and the ligated product
is reverse transcribed to generate cDNA. This reverse transcribed product
is combined with RNase H to digest at least some of the small RNA or
fragmented mRNA from the RNA/cDNA duplexes, generating amplification
templates. Those in the art will appreciate that the concentration of
unligated adaptors and adaptor by-products is also decreased during the
ribonuclease digestion process. At this point, reactions contain cDNA
copies of the RNA molecules in the sample. To meet the amplified product
input requirements for certain sequencing techniques, and in some
embodiments to append identifier sequences to the amplified products, the
reverse transcribed products may be amplified using appropriate primer
sets, wherein at least one forward primer, at least one reverse primer,
or both may comprise one or more identifier sequences, and a number of
PCR amplification cycles wherein detection is in a linear range when
plotted vs. cycle number (˜12-15 or ˜12-18 cycles of PCR).
Those in the art will appreciate that limiting the cycle number minimizes
the synthesis of spurious PCR products and preserves the integrity of the
RNA profile of the sample. In certain embodiments, at least one forward
primer, at least one reverse primer, or at least one forward and at least
one reverse primer comprise one or more identifier sequences. Certain
embodiments comprise use of ten sets of PCR primers that have the same
nucleotide sequence, except for a 6 bp "barcode" identifier sequence on
the 3' (reverse) primer that is specific to that reverse primer species.

[0192] In certain embodiments, the amplification reaction products are
subjected to size selection, for example but not limited to, gel
electrophoresis, to concentrate the amplified products in a desired size
range and remove PCR by-products. Appropriately size selected amplified
products can be used in the SOLiD® Sequencing System (Applied
Biosystems) workflow at the emulsion PCR (ePCR) step where the amplified
products are attached to beads, further amplified using ePCR and
ultimately sequenced, which allows the presence, absence, and/or quantity
of various RNA molecules in the sample to be determined.

[0193] Those in the art will appreciate that, in certain circumstances, an
amplified product and/or a ligated product can serve as a surrogate for
the corresponding RNA molecule and that by detecting the amplified
product, the ligated product, or both, the RNA molecule is indirectly
detected and that such detection is within the scope of the current
teachings.

[0194] Kits:

[0195] Kits for performing certain of the instant methods are also
disclosed. Certain kit embodiments include first adaptors, second
adaptors, a polypeptide comprising double-strand specific RNA ligase
activity, reverse transcriptase, ribonuclease H(RNase H), DNA polymerase,
primers, or combinations thereof. In some embodiments, kits further
comprise an agent for removing 5' phosphates from RNA, for example but
not limited to, tobacco acid pyrophosphatase.

[0196] The instant teachings also provide kits designed to expedite
performing certain of the disclosed methods. Kits may serve to expedite
the performance of certain disclosed methods by assembling two or more
components required for carrying out the methods. In certain embodiments,
kits contain components in pre-measured unit amounts to minimize the need
for measurements by end-users. In some embodiments, kits include
instructions for performing one or more of the disclosed methods. In some
embodiments, the kit components are optimized to operate in conjunction
with one another.

[0197] In certain embodiments, kits comprise at least one first adaptor
species, at least one second adaptor species, a polypeptide comprising
double-strand specific RNA ligase activity, a DNA polymerase, including
without limitation, a RNA-directed DNA polymerase, a DNA-directed DNA
polymerase, or a DNA polymerase comprising both RNA-directed and
DNA-directed DNA polymerase activities, ribonuclease H, or combinations
thereof. In certain embodiments, the ligase comprises bacteriophage T4
RNA ligase 2 (Rnl2) or a ligase from the Rnl2 family. In some
embodiments, the first adaptor, the second adaptor, or both the first
adaptor and the second adaptor comprise a single-stranded portion
comprising degenerate sequences.

[0198] In certain embodiments, a kit comprises a plurality of first
adaptor species, wherein each first adaptor species comprises a different
degenerate sequence, a plurality of second adaptor species, wherein each
second adaptor species comprises a different degenerate sequence, a
ligase of the Rnl2 family, a RNA-directed DNA polymerase, a plurality of
different first primer species, a DNA-directed DNA polymerase and RNase H
(EC 3.1.26.4). In some embodiments, the kit further comprises tobacco
acid pyrophosphatase.

[0199] In certain embodiments, a kit comprises a plurality of first
adaptor species, wherein at least some of the first adaptor species
comprise a degenerate sequence, a plurality of second adaptor species,
wherein at least some of the second adaptor species comprise a degenerate
sequence, a polypeptide comprising double-strand specific RNA ligase
activity, a DNA polymerase, at least one primer species, and a
ribonuclease. In some embodiments, the DNA polymerase of the kit
comprises an RNA-dependent DNA polymerase and a DNA-dependent DNA
polymerase. In addition, the kit may comprise tobacco acid
pyrophosphatase.

[0200] In certain embodiments, kits further comprise a forward
amplification primer and a reverse amplification primer. In some
embodiments, a forward primer, a reverse primer, or both a forward and a
reverse primer comprise a universal priming sequence or the complement of
a universal priming sequence. In some embodiments, kits comprise a
forward primer, a reverse primer, or a forward primer and a reverse
primer that further comprises a reporter group. In some such embodiments,
the reporter group of a forward primer of a primer pair is different from
the reporter group of the reverse primer of the primer pair. In some
embodiments, kits further comprise at least one of: a reporter probe, a
nucleic acid dye, a reporter group, or combinations thereof. In some
embodiments, kits further comprise a control sequence, for example but
not limited to an internal standard sequence such as a housekeeping gene
or a polynucleotide ladder comprising molecular size or weight standards.

[0201] In certain kit embodiments a first adaptor, a second adaptor, a
forward primer, a reverse primer, or combinations thereof, comprise an
identification sequence or the complement of an identification sequence.
In certain embodiments, the identification sequence comprises one of (i)
5'-AAGCCC, (ii) 5'-CACACC, (iii) 5'-CCCCTT, (iv) 5'-CATCGG, (v) 5-TCGTTG,
(vi) 5'-GGGCAC, (vii) 5'-CCAGAC, (viii) 5'-CTCCGT, (ix) 5'-CCCTTC, (x)
5'-GCGGTC, or the complement of any one of these sequences (i)-(x). In
some embodiments, a reverse primer comprises the sequence of one of SEQ
ID NO:6 and SEQ ID NO:15 to SEQ ID NO:23. In some kit embodiments,
mixtures of forward primers and reverse primers are provided. In some
embodiments, kits provide a plurality of primer mixtures, for example but
not limited to, at least two of: (i) a primer mixture comprising a first
forward primer and a first reverse primer, wherein the first reverse
primer comprises a first identification sequence; (ii) a primer mixture
comprising the first forward primer and a second reverse primer, wherein
the second reverse primer comprises a second identification sequence;
(iii) a primer mixture comprising the first forward primer and a third
reverse primer, wherein the third reverse primer comprises a third
identification sequence; (iv) a primer mixture comprising the first
forward primer and a fourth reverse primer, wherein the fourth reverse
primer comprises a fourth identification sequence; (v) a primer mixture
comprising the first forward primer and a fifth reverse primer, wherein
the fifth reverse primer comprises a fifth identification sequence; (vi)
a primer mixture comprising the first forward primer and a sixth reverse
primer, wherein the sixth reverse primer comprises a sixth identification
sequence; (vii) a primer mixture comprising first forward primer and a
seventh reverse primer, wherein the seventh reverse primer comprises a
seventh identification sequence; (viii) a primer mixture comprising first
forward primer and an eighth reverse primer, wherein the eighth reverse
primer comprises an eighth identification sequence; (ix) a primer mixture
comprising the first forward primer and a ninth reverse primer, wherein
the ninth reverse primer comprises a ninth identification sequence; and
(x) a primer mixture comprising the first forward primer and a tenth
reverse primer, wherein the tenth reverse primer comprises a tenth
identification sequence.

[0202] Some kit embodiments comprise at least one adaptor mix, wherein
each adaptor mix comprises a first adaptor and a second adaptor; at least
one polypeptide comprising double-strand specific RNA ligase activity; a
reverse transcriptase (RNA-directed DNA polymerase); a DNA polymerase
(DNA-directed DNA polymerase); a ribonuclease; a mixture of
deoxyribonucleotide triphosphates (dNTPs); and at least one amplification
primer mix, wherein each amplification primer mix comprises a forward
primer and reverse primer. In some embodiments, the RNA-directed DNA
polymerase and the DNA-directed DNA polymerase comprise either an
RNA-directed DNA polymerase that possesses DNA-directed DNA polymerase
activity under certain reaction conditions or a DNA-directed DNA
polymerase that possesses RNA-directed DNA polymerase activity under
certain reaction conditions, for example but not limited to, Tth DNA
polymerase and DNA polymerase I from Carboxydothermus hydrogenoformans.
In some kit embodiments, the DNA-dependent DNA polymerase comprises Taq
DNA polymerase, including enzymatically active mutants and variants
thereof, for example but not limited to, AmpliTaq® DNA polymerase and
AmpliTaq Gold® DNA polymerase (Applied Biosystems). In some
embodiments, the ribonuclease comprises ribonuclease H(RNase H). Some kit
embodiments comprise at least one control RNA molecule, for example but
not limited to, at least one positive control RNA molecule, at least one
negative control RNA molecule, or both.

[0203] Solid Supports:

[0204] In certain embodiments, the disclosed methods and kits comprise a
solid support. In some embodiments, a solid support is used in a
separating and/or detecting step, for example but not limited to, for
purifying and/or analyzing amplification products. Non-limiting examples
of solid supports include, agarose, sepharose, polystyrene,
polyacrylamide, glass, membranes, silica, semiconductor materials,
silicon, organic polymers; optically identifiable micro-cylinders;
biosensors comprising transducers; appropriately treated or coated
reaction vessels and surfaces, for example but not limited to, micro
centrifuge or reaction tubes, wells of a multiwell microplate, and glass,
quartz or plastic slides and/or cover slips; and beads, for example but
not limited to magnetic beads, paramagnetic beads, polymer beads,
metallic beads, dye-impregnated or labeled beads, coated beads, glass
beads, microspheres and nanospheres. Those in the art will appreciate
that any number of solid supports may be employed in the disclosed
methods and kits and that the shape and composition of the solid support
is generally not limiting.

[0205] The current teachings, having been described above, may be better
understood by reference to examples. The following examples are intended
for illustration purposes only, and should not be construed as limiting
the scope of the teachings herein in any way.

[0211] The lyophilized oligonucleotides were resuspended to a stock
concentration of 100 μM in nuclease-free water (Ambion P/N AM9937) and
diluted accordingly. The RNA mixture was heated at 65° C. for 10
minutes followed by 5 minutes at 16° C. to hybridize the adaptors
to the small RNA in the sample.

[0212] The hybridized RNA/Adaptors were then mixed with 10 μL Ligation
Buffer (100 mM Tris-HCl pH 7.5, 20 mM MgCl2, 20 mM dithiothreitol, 2
mM ATP, and 40% (w/v) PEG-8000) and ligated with 20 units of T4 RNA
ligase 2 (NEB, Ipswich, Mass.) in a total of 20 μL for 16 hours at
16° C. In addition, a minus ligase control was prepared for the
total RNA sample using nuclease-free water in place of the ligase enzyme.

[0213] The ligated products were reverse transcribed by incubating for 30
minutes at 42° C. with 200 units of ArrayScript® reverse
transcriptase (Ambion P/N AM2048), 4 μL 10×RT buffer (provided
with the ArrayScript® enzyme) and 2 μL 2.5 mM dNTP mix (Ambion P/N
AM8228G) in a total volume of 40 μL. A minus RT control was included
for the total RNA sample using nuclease-free water in place of the
reverse transcriptase.

[0214] Excess RNA byproducts were removed by incubating a 10 μL aliquot
of the sample at 37° C. for 30 minutes with 10 units of RNase H
(Ambion P/N AM2292) and the RNase-treated cDNA was amplified by PCR. The
50 μL PCR reactions contained the following:

[0215] The PCR conditions were as follows: initial denaturation at
95° C. for 5 minutes, followed by 15 cycles of 95° C. for
30 seconds (denaturation), 62° C. for 30 seconds (annealing), and
72° C. for 30 seconds (extension). A final extension at 72°
C. for 7 minutes followed.

[0216] Ten μL of each sample was mixed 1:1 with Gel Loading Buffer II
(Ambion P/N AM8547) and the entire volume was loaded onto a 1.0 mm 6%
polyacrylamide gel and electrophoresed for approximately 45 minutes at
180 volts constant in 1× tris-borate EDTA running buffer (Ambion
P/N AM9863). The gel was removed from the cassette and stained in
1×SYBR® Gold nucleic acid gel stain (Invitrogen P/N S11494) in
1×TBE for 5 minutes. The gel was imaged using the Alpha Innotech
Fluor Chem SP imager and the image processed with AlphaEase® FC
software version 6.0.0.

[0217] As seen in FIG. 5, the amplified ligation products (amplified
product) are shown by bracket A; arrow B denotes undesired by-products of
the reaction that were amplified in the amplification reaction; bracket C
indicates the residual unligated adaptors and primers in the
amplification reaction composition.

[0218] Note that the forward PCR primer sequence contains SEQ ID NO:1, the
sequence of the first oligonucleotide (with T substituted for U),
beginning at nucleotide 19 of the forward primer. Note also that the
reverse primer sequence contains SEQ ID NO:4, the sequence of the fourth
oligonucleotide (with T substituted for U), beginning at nucleotide 30 of
the reverse primer. Therefore, with this construction, a detected small
RNA will have a length equal to a gel fragment size as seen in FIG. 5,
for example, less the total of the lengths of the primers.

Example 2

Generation of Amplified Product Using Various Double-Strand Specific
Ligases

[0221] As seen in FIG. 6, the amplified ligation products (amplified
product) are shown by bracket A; arrow B denotes undesired by-products of
the reaction that were amplified; and bracket C indicates the residual
unligated adaptors and primers in the amplification reaction composition.
Surprisingly, when the amplified products generated using ligase Rnl2
(lane 2), ligase Rnl1 (lane 4), ligase Dnl (lane 6), or a combination of
both Rnl1 and Dnl (lane 8) are compared, a different amplified product
profile is observed. Amplified product in the 110 base pair (bp) to 130
bp size range (arrow A), which in this illustrative embodiment is in the
size range expected for amplified miRNA, is observed when Rnl2 ligase is
used, but not when Rnl1 or Dnl are used, either alone or in combination.
Additionally, a prominent band of amplified product of approximately 100
bp (migrating slightly above B in FIG. 6) appears in all lanes
corresponding to Rnl1, Dnl or both (with and without reverse
transcriptase), but this band is not observed when Rnl2 is used. This
difference in amplified products generated with Rnl2 in comparison to the
amplified products generated using two different ligases either alone or
in combination in parallel reactions is surprising and unexpected.

Example 3

Generation of Barcoded Amplified Products for Sequencing

[0222] Small RNA is obtained from HeLa cells using the mirVana® miRNA
Isolation Kit (AM1560, Ambion) according to the manufacturer's
Instruction Manual following the procedures for total RNA isolation.

[0226] To determine the appropriate number of PCR cycles to use with a
given solution comprising amplified product, a small scale (i.e., 50
μL) PCR reaction is recommended. Small scale amplification reaction
compositions were prepared by combining 49.5 μL of this PCR master mix
with 0.5 μL of the solution comprising amplification template in a
RNase-free 0.2 mL thin-walled PCR tube. The amplification reaction
compositions were placed in a thermal cycler with a heated lid, heated at
95° C. for five minutes, then cycled for 12-15 cycles using a
profile of 95° C. for 30 seconds-62° C. for 30
seconds-72° C. for 30 seconds; then a final extension step is
performed at 72° C. for 7 minutes. The optimum number of
amplification cycles depends on the amount of amplification product in
the initial amplification reaction composition. A 5-10 μL aliquot of
the amplification reaction composition comprising amplified product was
analyzed by electrophoresis using a 6% native tris-borate EDTA (TBE)
acrylamide gel to determine the optimum number of amplification cycles.
Following such determination, a large scale amplification is performed.

[0227] To generate sufficient quantities of amplified product for
sequencing and/or other downstream processes, a larger scale
amplification reaction was performed by PCR. Master mix was prepared by
combining, for each reaction to be performed, 77 μL NF water (Ambion
AM9922), 10× GeneAmp® PCR Buffer I (or 10×PCR Buffer I,
Applied Biosystems P/N N8080160), 2 μL barcoded PCR primers (mix of
forward and a barcoded reverse primer of choice), 8 μL dNTP mix, and 2
μL AmpliTaq® DNA-directed DNA Polymerase (Applied Biosystems P/N
N8080160). A 99 μL aliquot of this master mix and 1 μL of the
solution comprising amplification templates were combined in three
separate wells of an RNase-free PCR plate (i.e., in triplicate) to form
an amplification reaction composition. The PCR plate was heated to
95° C. for 5 minutes to denature the nucleic acid, cycled
according to the previous temperature profile (95° C. for 30
seconds-62° C. for 30 seconds-72° C. for 30 seconds) for
the previously determined number of cycles and finally the PCR reaction
vessel was maintained at 72° C. for 7 minutes to generate
amplified products in the amplification reaction composition. The
amplified products were pooled analyzed by electrophoresing 5-10 μL of
the pooled amplification reaction compositions on a 6% native TBE
acrylamide gel as before.

[0228] Two hundred and fifty (250) μL of the pooled amplified product
was combined with 250 μL phenol/chloroform/isoamyl alcohol (25:24:1,
pH 7.9) in an RNase-free 1.5 mL polypropylene microfuge tube and mixed by
vortexing. The tube was centrifuged at 12,000 rpm for 5 minutes at room
temperature using a benchtop centrifuge. The aqueous phase was measured
and transferred to a fresh RNase-free 1.5 ml polypropylene microfuge tube
and an equal volume of 7.5 M ammonium acetate added to the tube along
with 1/100 volume of glycogen (or GlycoBlue® Co-precipitant (Ambion)
and 0.7 volumes isopropanol. The contents of the tube are mixed
thoroughly, incubated at room temperature for 5 minutes, and then
centrifuged at 12,000 rpm for 20 minutes at room temperature. The
resulting supernatant was removed and discarded and the pellet washed
three times with 1 mL of 70% (v/v) ethanol. The pellet was air dried,
then resuspended in 18 μL nuclease-free water to which 2 μL of
10× native gel loading dye is added. Ten μL of this suspension
was added to each of two wells of a native TBE PAGE gel which contains a
10 basepair (bp) molecular weight ladder (Invitrogen 10821-015) as a
marker in one of the other wells. The amplified products were
electrophoresed in the gel at ˜140 V until the dye front is about
to elute off the bottom edge of the gel (˜30 minutes for a 1.0 mm,
8 cm×8 cm gel). The nucleic acid bands in the gel were stained
using SYBR® Gold (Invitrogen, Carlsbad, Calif.) following the
manufacturer's instructions and illuminated using an ultraviolet light
source. Using a clean razor blade, the gel was sliced in the lanes
containing amplified product to obtain the nucleic acid in the size range
of approximately 100 to 150 bp. A distinct band at 100 bp likely
represents undesired byproducts and was not included with the slice
excised from the gel. Likewise, nucleic acid larger than about 200 bp was
also avoided when certain sequencing methods were employed, for example
but not limited to, emulsion PCR sequencing using the SOLiD® System
(Applied Biosystems).

[0229] A hole was made in the bottom of an RNase-free 0.5 mL polypropylene
microfuge tube using a 21 gauge needle and the excised gel piece is
transferred to the tube. This 0.5 mL tube was then placed inside an
RNase-free 1.5 mL polypropylene microfuge tube and centrifuged for 3 min
at 12,000 rpm to shred the gel. The 0.5 mL tube is removed and discarded
and the outer 1.5 mL tube containing the gel fragments is placed on ice.
Two hundred (200) μL PAGE elution buffer (1.5 M ammonium acetate in
1×TE buffer pH 7.0) was added to tube, which was then incubated at
room temperature for 20 minutes. After this first incubation, the
supernatant was removed and transferred to a clean RNase-free 1.5 mL
polypropylene microfuge tube and an additional 250 μl of PAGE elution
buffer was added to the first tube (containing the gel fragments) and
incubated for an additional 40 minutes at 37° C. Following the
second incubation, the second supernatant was collected and added to the
first. Residual gel pieces were removed form the pooled supernatants
using a spin column (Ambion Cat#10065) and centrifugation, according to
the manufacturer's instructions.

[0230] The resulting liquid was combined with an equal volume of
phenol/chloroform/isoamyl alcohol (25:24:1, pH 7.9) in an RNase-free 1.5
mL polypropylene microfuge tube and mixed by vortexing. The tube was
centrifuged at 12,000 rpm for 5 minutes at room temperature using a
benchtop centrifuge. The aqueous phase was measured and transferred to a
fresh RNase-free 1.5 ml polypropylene microfuge tube and an equal volume
of 7.5 M ammonium acetate was added to the tube along with 1/100 volume
of glycogen (AM9510, Ambion; or GlycoBlue® Co-precipitant AM9515,
Ambion) and 0.7 volumes isopropanol. The contents of the tube were mixed
thoroughly, incubated at room temperature for 5 minutes, and then
centrifuged at 12,000 rpm for 20 minutes at room temperature. The
resulting supernatant was removed and discarded and the pellet washed
three times with 1 mL of 70% (v/v) ethanol. The pellet was air dried,
then resuspended in 204 NF water. The DNA comprising the amplified
product was quantitated by determining the A260 with a
spectrophotometer or by analyzing on a 6% native PAGE gel, as described
above.

[0231] To determine the sequence of the amplified product, emulsion PCR
(ePCR) was performed on an Applied Biosystems SOLiD® System according
to the User Guide (Applied Biosystems, P/N4391578; the "User Guide"). To
evaluate what concentration of amplified product that gives the best
sequencing results in a full scale ePCR on the SOLiD® System, four
separate ePCR reactions were performed at amplified product
concentrations of 0.2 pg/μL, 0.4 pg/μL, 0.6 pg/μL, and 0.8
pg/μL, followed by a titration/QC run according to the manufacturer's
instructions (see particularly, the User Guide, Chapters 3 and 4). When
the optimal amplified product concentration was determined, a "full
scale" ePCR reaction was performed (see Section 3.1, Chapter 3 of the
User Guide). By determining the sequence of at least part of the
amplified product, one can directly or bioinformatically identify the RNA
molecule from which that amplified product was derived, thereby detecting
that RNA molecule. Those in the art will appreciate that such sequence
information may be used to identify novel RNA molecules, including
without limitation, small RNA discovery; may be used to quantitate the
amount of one detected RNA molecule species in the starting sample
relative to the amount another detected RNA species and such information
may be useful for, among other things, expression profiling of mRNA,
miRNA or other RNA molecules of interest.

Example 4

Evaluation of Adaptor Overhang Length

[0232] First and second adaptors with various overhang lengths were
synthesized and evaluated in an effort to maximize ligation efficiency
while minimizing adaptor complexity.

[0233] Exemplary first adaptors comprised first oligonucleotides, depicted
as "T3" in FIG. 7B wherein the first oligonucleotides comprised a DNA
sequence from bacteriophage T3 and two ribonucleotides at the 3' end, and
second oligonucleotides, depicted as "27 N" in FIG. 7B wherein the second
oligonucleotides comprised a complementary deoxyribonucleotide sequence
from bacteriophage T3 and an overhang of 4, 6 or 8 degenerate
deoxyribonucleotides "N" at the 5' end. The upper strand (first
oligonucleotide) of an illustrative first adaptor comprised a 27
nucleotide sequence from the bacteriophage T3 promoter
5'-CUCGAGAAUUAACCCUCACUAAAGGGA-3' (SEQ ID NO:7), shown as "T3" in FIG.
7B. The lower strand (second oligonucleotide) comprised the complementary
sequence of the upper strand with either 4, 6, or 8 degenerate
nucleotides (depicted as "N" for illustration purposes in FIG. 7B) on the
5' end of the lower strand 5-(N)4,6,8TCCCTTTAGTGA GGGTTAATTCTCGAG-3'
(SEQ ID NO:8 (where N=8); SEQ ID NO:8 lacking either 2 or 4 5'-N's (i.e.,
where N is 6 or 4)), depicted as "27 N" for illustration purposes in FIG.
7B.

[0234] Exemplary second adaptors comprised third oligonucleotides,
depicted as "T7" in FIG. 7B wherein the third oligonucleotides comprised
a DNA sequence from bacteriophage T7, and fourth oligonucleotides,
depicted as "N 28" in FIG. 7B wherein the fourth oligonucleotides
comprised a complementary deoxyribonucleotide sequence from bacteriophage
T7 and an overhang of 4, 6 or 8 degenerate deoxyribonucleotides "N" at
its 3' end. The upper strand (third oligonucleotide of an illustrative
second adaptor comprised a 28 nucleotide sequence from the bacteriophage
T7 promoter 5'-PO4-TCCCTATAGTGAGTCGTATTACGAATTC-3'(SEQ ID NO:9)
shown as "T7" in FIG. 7B which comprises a 5' phosphate group (shown as
PO4). The lower strand (fourth oligonucleotide) comprised the
complementary sequence of the upper strand with either 4, 6, or 8
degenerate nucleotides (depicted as "N" for illustration purposes in FIG.
7B) on the 3' end of the lower strand
5'-GAATTCGTAATACGACTCACTATAGGGA(N)4,6,8-3' (SEQ ID NO:10 (where
N=8); SEQ ID NO:10 lacking either 2 or 4 3'-N's (i.e., where N is 6 or
4)), depicted as "N 28" for illustration purposes in FIG. 7B.

[0235] Fifty picomoles (pmol) of either the "T3" first adaptors (see FIG.
7B), the "T7" second adaptors (see FIG. 7B), or both the T3 first
adaptors and the T7 second adaptors (both with the same number of
degenerate nucleotides) in 2 μL water was incubated with 3 μL
2× Hybridization Buffer (300 mM NaCl, 20 mM Tris pH8, 2 mM EDTA) at
95° C. for 3 minutes, then cooled to 22° C. When the
temperature reached 22° C., 1 μL of a 0.13 μM 5'
32P-labeled synthetic microRNA pool (mirVana® miRNA Reference
Panel 9.0, a pool of approximately 500 synthetic miRNA sequences from the
Sanger miRBase 9.0 database (microrna.sanger.ac.uk/sequences/) in
equimolar concentration) was added to each reaction tube, the tubes were
incubated at 65° C. for 10 minutes, then cooled down to 22°
C.

[0237] Different structures of 5' (first) and 3' (second) adaptors with
6-nucleotide degenerate overhangs were tested which included dsDNA (with
the exception of two 3' terminal RNA bases on the upper strand (first
oligonucleotide) of the 5' (first) adaptor (e.g., T3r2:27 6N and T7:6 N
28 in FIG. 8B (the numbers 27 and 28 refer to the length of the first and
third oligonucleotides, respectively, as in Example 4) and
dsRNA•DNA hybrid (upper (first and third oligonucleotides are
RNA)-lower (second and fourth oligonucleotides are DNA) for both adaptors
(e.g., rT3:27 6N and rT7:6 N 28 in FIG. 8B).

[0238] In this illustrative embodiment of FIG. 8B, top first adaptor
(rT3:27 6N) comprises an upper strand (first oligonucleotide) comprising
the T3 sequence (SEQ ID NO:7) wherein all of the nucleosides comprise
ribonucleosides, shown as "rT3," annealed to the lower strand (second
oligonucleotide) comprising the complementary sequence and 6 degenerate
nucleotides on the 5' end of the lower strand (SEQ ID NO:8 where N is 6),
depicted as "27 6N" for illustration purposes; and the other illustrative
first adaptor (T3r2:27 6N) comprises an upper strand comprising the T3
sequence wherein all of the nucleosides comprise deoxyribonucleosides
except for the two 3'-most nucleosides which comprise ribose (SEQ ID NO:7
having 2 3'-terminal ribonucleotides), shown as "T3r2" annealed to the
same lower strand as the top first adaptor (SEQ ID NO:8 where N is 6)
depicted as 27 6N.

[0239] Again referring to FIG. 8B, a top illustrative second adaptor
(rT7:6 N 28) comprises an upper strand (third oligonucleotide) comprising
the T7 sequence (SEQ ID NO:9) wherein all of the nucleosides comprise
ribonucleosides and which sequence comprises a 5' phosphate group,
depicted as "rT7" for illustration purposes, annealed to the lower strand
which comprises the complementary sequence with 6 degenerate nucleotides
on the 5' end of the lower strand (SEQ ID NO:10 where N is 6), depicted
as "6N 28" for illustration purposes. The other illustrative second
adaptor (T7:6 N 28) comprises an upper strand comprising the T7 sequence
(SEQ ID NO:9) wherein all of the nucleosides comprise
deoxyribonucleosides and which sequence comprises a 5' phosphate group,
depicted as "T7" for illustration purposes, annealed to the same lower
strand as the top second adaptor (SEQ ID NO:10 where N is 6), depicted as
6N 28.

[0240] Fifty pmol of either the first adaptors, the second adaptors, or
both the first adaptors and the second adaptors, in 2 μL water was
incubated with 3 μL 2× Hybridization Buffer (300 mM NaCl, 20 mM
Tris pH8, 2 mM EDTA) at 95° C. for 3 minutes and cooled down to
22° C. One (1) μL of a 0.13 μM 5' 32P-labeled miRNA
pool (mirVana® miRNA Reference Panel as cited above) was added to each
reaction, then incubated at 65° C. for 10 minutes, and cooled down
to 22° C.

[0242] Three different combinations of first adaptors and second adaptors
were tested for double ligation efficiency. These combinations included
first adaptors and second adaptors with both DNA upper strands (i.e.,
first and third oligonucleotides are DNA with the exception that the
first oligonucleotide has two 3' ribonucleotides), both RNA upper strands
(i.e., first and third oligonucleotides), or RNA upper strand on 5'
(first) adaptor (i.e., first oligonucleotide) and DNA upper strand on 3'
(second) adaptor (i.e., third oligonucleotide) (see FIG. 9c for a
schematic of the latter adaptor structure embodiment). One (1) μL of
each adaptor (50 μM each) was combined and incubated with 3 μL
2× Hybridization Buffer (300 mM NaCl, 20 mM Tris pH8, 2 mM EDTA) at
95° C. for 3 minutes and cooled down to 22° C. One (1)
μL of a 0.13 μM 5' 32P-labeled synthetic miRNA pool (cited
above) was added to each 5 μL reaction, the reactions were then
incubated at 65° C. for 10 minutes and then cooled down to
22° C.

[0247] For the samples of lanes 7-12 shown on the gel in FIG. 10A, an
RNase H digestion reaction was performed as follows followed by PCR. Five
μL of the RT reaction was transferred to a clean tube, mixed with 0.5
μL Ribonuclease H(RNase H, 10 U/μL, Ambion AM2292) and incubated at
37° C. for 30 minutes. One (1) μL of RNase H treated sample was
used for PCR reaction at the same condition as described previously. All
of these amplified products (both with and without RNase H digestion)
were loaded on a 10% native PAGE gel, electrophoresed, and visualized by
SYBR® Gold (Invitrogen 11494) staining, as shown in FIG. 10A.

[0248] Adaptors with different upper/lower strand ratios as indicated in
the table shown in FIG. 10B were mixed with 2 pmol synthetic mirVana®
miRNA Reference Panel (cited above) and 3 μL 2× Hybridization
Buffer (300 mM NaCl, 20 mM Tris pH8, 2 mM EDTA) in a 6 μL reaction
mixture followed by incubating at 65° C. for 15 minutes and
16° C. for 1 hour. The ligation, RT and RNase H treatment were
performed as described previously. One (1) μL of RNase H treated
sample was used for PCR amplification with SuperTaq® polymerase
(Ambion AM2050) in 20 cycles as described before. Five μL of each of
the PCR products were loaded on a 10% native PAGE gel, electrophoresed,
and visualized by SYBR® Gold (Invitrogen 11494) staining, as shown in
FIG. 10B. Adaptors with picomolar ratios of upper to lower strand of
1/50, 5/50 10/50, 25/50 and 5/100 were all competent to generate the
desired products migrating at >50 bp. In contrast, use of an adaptor
ratio of 5/500 did not efficiently generate the desired products.

Example 7

Comparison of the Present Method with TaqMan® miRNA Assays

Samples that Vary in RNA Content

[0249] The present example provides a quantitative validation of the
present methods as compared to the RT-PCR TaqMan® miRNA assays. FIG.
12 depicts a scatter plot depicting a relative fold change (FC)
comparison of miRNA quantitation results obtained from human placental
RNA and from human lung RNA using 5' nuclease assays with sequencing
results obtained according to certain embodiments of the current
teachings. The x-axis shows the log2 fold change of 5' nuclease
assay results in -ΔΔCT ((generated using TaqMan® Human
MicroRNA Array v1.0 (P/N 4384792; Applied Biosystems) and Multiplex RT
for TaqMan® MicroRNA Assays (P/Ns 4383403, 4383402, 4383401, 4383399,
4384791, 4382898, 4383405, 4383400, and 4383404; Applied Biosystems)
performed essentially according to the manufacturer's protocol) and the
y-axis shows the log2 fold change of sequencing data (generated
using the SOLiD® Sequencing System (Applied Biosystems) essentially
according to the manufacturer's protocol) according to an embodiment of
the current teachings. TaqMan® MicroRNA Assays (Applied Biosystems)
that generated CT values above 35 were presumed to be negative and
not included in the analysis. For SOLiD® sequencing data, data from at
least 3 sequenced tags were required for the corresponding sequence to be
considered as `observed.` Using this approach an R value of 0.88 was
obtained.

[0253] Then 5 μL of RT reaction was transferred to a clean tube, mixed
with 0.5 μL Ribonuclease H(RNase H, 10 U/μL, Ambion AM2292) and
incubated at 37° C. for 30 minutes. 0.5 μL of RNase H treated
sample was then combined with 5 μL 10× AmpliTaq® Buffer I
(Applied Biosystems N8080171), 4 μL dNTP (2.5 mM), 0.5 μL 5' primer
and 0.5 μL 3' primer (50 μM each), 1 μL of AmpliTaq® DNA
polymerase (5 U/μL, Applied Biosystems N8080171) and 38.5 μL of
RNase-free water. PCR was performed using 16 cycles of 30 seconds at
95° C., 30 seconds at 62° C. and 30 seconds at 72°
C. Five (5) μL of PCR products were loaded on a 10% native PAGE gel
and visualized by SYBR® Gold (Invitrogen 11494) staining. As shown by
FIG. 13A, note that the placenta sample is fairly rich in small RNAs and
the method presented herein is capable of producing small RNA products
from ≦25 ng total RNA. In contrast, the mouse liver sample as
shown by FIG. 13B is very poor in small RNAs by comparison and thus,
according to certain embodiments of the current teachings, enrichment of
such samples may provide better results.

[0254] Sequences for the adaptor Mix A are as for SEQ ID NO:1-SEQ ID NO:4
of Example 1.

[0255] Four synthetic RNA oligonucleotides containing 5'-PO4 (spike in
controls, SIC; sequences shown below) were mixed at varying concentration
spanning a 1000-fold input range (1000, 100, 10, and 1 pg in 1000×
mix), and the mixture was serially diluted to 1×. Mixtures were
spiked into 500 ng placenta total RNA as background (FIG. 14, FIG. 16A)
and the ligation reaction was performed on the four samples as described
previously except that the ligation reaction composition was incubated
for two hours (instead of sixteen hours) at 16° C.

[0260] Following the ligation reaction a mixture containing appropriate
buffer, dNTPs, ArrayScript® reverse transcriptase, AmpliTaq® DNA
polymerase and both forward and reverse PCR primers are added directly to
the ligation reaction and mixed by gentle pipetting up and down several
times (3-4×). The reaction mixture is then incubated at 42°
C. for 30 minutes in a thermal cycler to permit reverse transcription to
generate an amplification template. RNase H is then added to the reaction
mixture and incubated at 37° C. for 30 minutes in a thermal
cycler. The reaction temperature is then ramped up to 95° C. and
held for 5 minutes followed by standard amplification cycles, i.e., 30
seconds at 62° C. and 30 seconds at 72° C., as described in
Example 1 to generate amplified products.

Example 10

Generating Amplification Templates and Amplified Products with One DNA
Polymerase

[0261] Following the ligation reaction a mixture containing appropriate
buffer (containing optimized concentrations of both manganese and
magnesium), dNTPs, a DNA polymerase comprising both DNA dependent DNA
polymerase activity and RNA dependent DNA polymerase activity and both
forward and reverse PCR primers is added to the ligation reaction
composition and mixed by gentle pipetting up and down several times
(3-4×) to from a reverse transcription composition. The reverse
transcription composition is incubated at 42° C. for 30 minutes in
a thermal cycler to permit the reverse transcriptase activity to generate
an amplification template. RNase H is then added to the reaction mixture
and incubated at 37° C. for 30 minutes in a thermal cycler. The
reaction temperature is then ramped up to 95° C. and held for 5
minutes followed by standard amplification cycles, i.e., 30 seconds at
62° C. and 30 seconds at 72° C., as described in Example 1
to generate amplified products.

Example 11

Exemplary Method for Generating a Small RNA Library

[0262] The present example provides exemplary methods for generating a
small RNA library as depicted in FIG. 17. When the RNA molecule of
interest is a small RNA, the starting material should comprise the small
RNA fraction. FirstChoice® prepared Total RNA (Applied Biosystems) is
certified to contain miRNA and other small RNAs. Alternatively, total RNA
that includes the small RNA fraction of a sample may be obtained using
the mirVana® miRNA Isolation Kit or mirVana® PARIS® Kit
according to user's manual following the procedures for total RNA
isolation.

[0263] Since RNA samples can vary widely in small RNA content based on
their source and the RNA isolation method, evaluating the small RNA
content of samples to determine whether to use total RNA or size-selected
RNA in reactions may be desirable, using for example, but not limited to,
an Agilent bioanalyzer with the Small RNA Chip.

[0264] Total RNA samples that contain more than 0.5% small RNA (in the
˜10-40 nt size range) can be used without size-selection. When
total RNA is used in the procedure the resulting reaction products will
be a larger size range than those produced from PAGE-purified small RNA
samples. In addition, SOLiD® sequencing results from total RNA samples
will typically include a slightly higher number of rRNA and tRNA reads.

[0265] RNA samples that contain less than 0.5% small RNA content should be
enriched for the ˜18-40 nt RNA fraction, for example but not
limited to, by PAGE and elution, by the flashPAGE® Fractionator and
flashPAGE® Reaction Clean-Up Kit (Applied Biosystems).

[0266] The relative amount of small RNA in different sample types varies
greatly as described in Example 7. For example, RNA from tissue samples
typically has a rich supply of small RNAs, whereas RNA from cultured cell
lines often has very few small RNAs.

[0269] Hybridization and Ligation are carried out as follows. An adaptor
mix A is designed for SOLiD® sequencing from the 5' ends of small
RNAs, for example, and an adaptor mix B is designed for SOLiD®
sequencing from the 3' ends. To sequence the small RNA in a sample from
both the 5' and 3' ends, two ligations were set up, one with each adaptor
mix. Each adaptor mix contains first, second, third and fourth
oligonucleotides. Adaptor mix B is in the reverse complement orientation
as compared to adaptor mix A so that each strand of an amplified product
can be detected. On ice, the hybridization mix is prepared in 0.2 mL PCR
tubes as follows.

[0270] The contents were mixed well by gently pipetting up and down a few
times, then centrifuged briefly to collect the solution at the bottom of
the tube. The reactions were placed in a thermal cycler with a heated
lid, programmed as follows.

[0272] The mix was incubated for 16 hours in a thermal cycler set to
16° C. A 2 hour incubation is generally sufficient for ligation,
however, an overnight incubation resulted in slightly higher amounts of
ligated product.

[0273] Reverse Transcription and RNase H Digestion:

[0274] The sample(s) were placed on ice and a Reverse Transcription (RT)
Master Mix was prepared on ice by combining the following reagents. An
extra 5-10% volume was included in the master mix to compensate for
pipetting errors.

[0275] RT Master Mix (20 μL) was added to each sample and the samples
were vortexed gently to mix thoroughly and microcentrifuged briefly to
collect the mixture at the bottom of the tube. The samples were then
incubated at 42° C. for 30 minutes to synthesize cDNA.

[0276] The cDNA can be stored at -20 C for a few weeks, at -80° C.
for long term storage, or used immediately in the RNase H digestion
(next).

[0277] RNase H incubation was carried out as follows. A volume (10 μL)
of the RT reaction mixture was transferred from the previous step (cDNA)
to a fresh tube. RNase H (1 μL) was added. The mixture was vortexed
gently to mix, microcentrifuged briefly to collect the mixture at the
bottom of the tube, and incubated at 37° C. for 30 minutes.

[0278] After the RNase H treatment, samples can be stored at -20°
C. overnight or used immediately in the PCR.

[0279] Small RNA Library Amplification:

[0280] Pilot and Large Scale PCRs: Because different sample types can
contain substantially different amounts of small RNA, the number of PCR
cycles needed to obtain enough DNA for SOLiD® sequencing also varies.
A 50 μL trial PCR was performed to determine the number of PCR cycles
needed for a given sample type before proceeding to a set of three or
more replicate 100 μL reactions (Large Scale PCRs) used to synthesize
template for the next step in SOLiD® sequencing sample preparation.

[0281] Most samples should be amplified for 12-15 cycles. For pilot
experiments, 12 PCR cycles are recommended for samples from starting
material with a relatively high amount of small RNA (i.e., total RNA from
tissue of ˜200-500 ng, or ˜50-200 ng size-selected small RNA)
and 15 cycles for those with relatively little small RNA (i.e., total RNA
from tissue of ˜1-200 ng, or total RNA from cultured cells of
˜100-500 ng, or 1-50 ng size-selected small RNA.

[0282] Small RNA PCR Primer Sets:

[0283] Ten different PCR primer sets for synthesis of SOLiD® sequencing
template are provided with the SOLiD® Small RNA Expression Kit
(Applied Biosystems). The primer sets are identical except for a 6 bp
barcode located near the middle of the primers. This barcode feature of
the PCR primers enables sequencing and analysis of multiplexed samples.
That is, it is possible to sequence up to ten different samples, one
amplified with each of the supplied SOLiD® Small RNA PCR Primer Sets,
in a single SOLiD® sequencing reaction. Any of the primer sets can be
used but samples are not mixed at this point.

[0286] The mix was vortexed gently to mix thoroughly and microcentrifuged
briefly to collect the mixture at the bottom of the tube. (Once the
appropriate number of PCR cycles for the sample type was determined, 3 or
more replicate large scale PCRs are run for each sample. Reaction
products are pooled to generate enough material for gel purification and
subsequent SOLiD® sequencing sample preparation.)

[0287] PCR Master Mix for a single reaction was pipetted into wells of a
PCR plate or 0.2 mL PCR tubes. For a trial PCR (50 μL), 0.5 μL
RNase H-treated cDNA was added to each aliquot of PCR Master Mix. For
large scale PCR (100 μL), 1 μL RNase H-treated cDNA was added to
each aliquot of PCR Master Mix. Greater than 1 μL cDNA in a 50 μL
PCR is not recommended due to possible reaction inhibition.

[0288] Sample(s) were placed in a thermal cycler with a heated lid and the
thermal profile shown below was carried out.

[0289] PCR product (5-10 μL) was run on a native 6% polyacrylamide gel
and the gel is stained with SYBR® Gold following the manufacturer's
instructions.

[0290] FIG. 5 shows results from reactions that were amplified using an
appropriate number of PCR cycles. Results are discussed in Example 1 and
generalized here for illustration.

[0291] The amplified product derived from small RNA migrates at
˜108-130 bp (the total length of the primers is ˜89 bp,
therefore, RNA of about 19-41 bp migrate in the cited range).

[0292] Note that higher molecular weight bands at approximately 150 and
200 bp are expected from reactions using total RNA as input, whereas
these larger products are not expected from reactions using size-selected
small RNA as input (see FIG. 5).

[0293] Self-ligated adaptors and their amplified products form a band at
89 bp. This band is typically present in all reactions. Further
by-products migrate at ˜100 bp. Underamplified samples exhibit very
little material in the ˜108-130 bp size range. Conversely,
overamplified samples typically show a significant amount of material in
the ˜108-130 bp size range, plus a smear of reaction products
larger than ˜140-150 bp. Overamplified samples from total RNA input
may also have a higher molecular weight ladder of bands that represent
concatenated PCR products.

[0294] Amplified Small RNA Library Cleanup:

[0295] The PCR products derived from small RNA were then cut from the gel,
eluted out of the acrylamide, purified, and concentrated as follows.
Samples were not heated at any step of this purification so that the DNA
duplexes remain annealed and migrate according to their size during
subsequent gel purification.

[0296] An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1, pH
7.9) was added to each sample. Samples were vortexed to mix, then
centrifuged at 13,000×g for 5 min at room temperature. The aqueous
(upper) phase was transferred to a fresh 1.5 mL tube, with the volume
measured during transfer. An equal volume of 5 M ammonium acetate was
added to each sample. An amount (1/100 volume) of glycogen and 0.7 volume
isopropanol were added (the sample volume after addition of ammonium
acetate is used as a baseline). The mixture was mixed thoroughly,
incubated at room temperature for 5 minutes, and centrifuged at
13,000×g for 20 minutes at room temperature. The supernatant was
carefully removed and discarded. The DNA pellet was washed 3 times with 1
mL of 70% ethanol each time and allowed to air dry for ˜15 minutes
or until visible droplets of ethanol had evaporated.

[0298] A needle (e.g., 21-gauge) was used to poke a hole through the
bottom-center of 0.5 mL microcentrifuge tube for each sample. The gel
pieces excised above were placed in these tubes, and the centrifugation
in the subsequent step shred the DNA-containing gel pieces for elution of
the DNA. The DNA pellet from above in 20 μL 1× nondenaturing gel
loading buffer was loaded onto a 6% native TBE polyacrylamide gel. A DNA
Ladder, or a similar ladder, was loaded in a separate lane as a marker.
The gel was run at ˜140 V (˜30 minutes for a minigel) or
until the leading dye front almost exits the gel and stained with
SYBR® Gold following the manufacturer's instructions. The gel piece
containing 105-150 bp DNA was excised using a clean razor blade and
placed in a 0.5 mL tube prepared with a hole in the bottom, the 0.5 mL
tube placed within a larger 1.5 mL tube. The gel piece was shredded by
microcentrifuging for 3 min at 13,000×g. An amount (200 μL) PAGE
elution buffer was added to the shredded gel pieces; the mixture
incubated at room temperature for 1 hour, and the buffer was transferred
to a fresh tube, leaving the gel fragments behind. The shredded gel
pieces were extracted again, this time at 37° C. and the elution
buffers were combined. Transfer the mixture to a Spin Column and
centrifuged at top speed for 5 minutes to remove gel pieces. The DNA was
in the flow-through.

[0299] An amount (1/100 volume) of glycogen and 0.7 volume isopropanol
were added to each sample. The samples were mixed thoroughly, incubated
at room temperature for 5 minutes, and centrifuged at 13,000×g for
20 min at room temperature. The supernatant was carefully removed and
discarded and the pellet was air dried, then resuspended in 20 μL
nuclease-free water.

[0300] DNA in each sample was quantitated by measuring the A260 in a
spectrophotometer (1 A260=50 μg DNA/mL) and verifying the size
and quality using an Agilent bioanalyzer or 6% native PAGE. The minimum
amount of DNA that can be used for SOLiD® sequencing is 200 ng at 20
ng/L, but more DNA is preferable. SOLiD® Small RNA Expression Kit
reaction products enter the SOLiD® sample preparation workflow at the
"SOLiD® System Template Bead Preparation" stage, in which emulsion PCR
is used to attach molecules to beads. SOLiD® sequencing and emulsion
PCR is described, for example, in published PCT applications WO 06/084132
entitled "Reagents, Methods, and Libraries For Bead-Based Sequencing and
WO 07/121,489 entitled "Reagents, Methods, and Libraries for Gel-Free
Bead-Based Sequencing."

Example 12

Validation of Present Methods for Mapping and Transcript Coverage

[0301] The present example provides for validation of present methods by
demonstrating that the methods are useful for mapping short (25-70 base)
tags of sequences to the human genome or other databases and provides
uniform transcript coverage.

[0302] For example, at least 15 libraries testing different conditions
were separately made, pooled and then sequenced in a single run. A
relatively equal number of miRNA sequences were found across the
libraries; any deviations from equal numbers detected likely represent
pipetting errors in the pooling step. In a single sample, 389 out of 555
miRNAs were detected (70%). Undetected miRNAs predominantly represent RNA
molecules that are not present in the sample.

[0303] Sequence reads cs.fasta files generated on the SOLiD® instrument
were mapped to databases of miRNAs, tRNAs, rRNAs, Refseqs and the genome.
Out of 17.7 million reads, 55% were mapped to the genome, 12% to RefSeq
sequences, 1% to rRNA, 0.5% to tRNA, 8.25% to miRNA and 23% were not
mapped. Reads that mapped to the genome uniquely were found to be
clustered, i.e., ˜3000 have at least two reads and a size of <70
bases. These sequences are candidates for novel miRNAs or ncRNAs.

[0304] An analysis of transcriptome sequencing using the SOLiD® System
of fifteen barcoded and pooled libraries found that, out of about 59.16
million reads, 36.22% were mapped to the genome, 6.98% to RefSeq
sequences, 0.32% to rRNA, and 0.01% to tRNA.

[0305] RNA detection was demonstrated to be reproducible. Placenta poly(A)
RNase III fragmented libraries generated under similar but not identical
conditions were found to have good reproducibility (R2 values of 0.97)
and a dynamic range of detection spanning at least 5 logs.

[0306] A whole transcript analysis was carried out using the SOLiD®
System and the coverage for mRNA to brain-specific angiogenesis inhibitor
2 (BAI2) was found to be uniform from 5' to 3'. Overlapping individual 50
base tags representing nearly the entire length of the mRNA were
observed. Specific tags represented multiple times can be a reflection of
the relative amount of the mRNA contained within the sample (that is, the
more RNA molecules present in the sample the higher the probability of
capturing the same tag of sequence), bias in the cleavage site of the
fragmented RNA, or possibly PCR amplification artifacts.

[0307] The density of starting points for sequencing tags was plotted
relative to the length of the RNA transcripts analyzed. The results
indicated that tag density is uniform across the body of the transcripts
and there is a drop off at the extreme 5' and 3' ends. This decrease in
capture tags at the ends of the RNA represent the inefficiency of
ligating to the 5' cap contained on all RNA polymerase II generated RNA
transcripts. Regarding the 3' end of mRNAs that typically contain a polyA
tail, the ability to capture and amplify this material appears hindered
and the proportion of tags is low, possibly due to the homopolymer nature
of the tail.

[0308] The compositions, methods, and kits of the current teachings have
been described broadly and generically herein. Each of the narrower
species and sub-generic groupings falling within the generic disclosure
also form part of the current teachings. This includes the generic
description of the current teachings with a proviso or negative
limitation removing any subject matter from the genus, regardless of
whether or not the excised material is specifically recited herein.

[0309] Although the disclosed teachings have been described with reference
to various applications, methods, and compositions, it will be
appreciated that various changes and modifications may be made without
departing from the teachings herein. The foregoing examples are provided
to better illustrate the present teachings and are not intended to limit
the scope of the teachings herein. Certain aspects of the present
teachings may be further understood in light of the following claims.